Method for screening drug for improving insulin resistance

ABSTRACT

Disclosing a method for screening a protein interactive with PPAR in a ligand-dependent manner, works as a useful tool for screening a drug ameliorating insulin resistance. By the method, ECHLP as a main action ligand-dependent PPAR binding molecule, FLJ13111 as a main action ligand-selective factor interactive with PPARγ and AOP2 as an adverse action ligand-dependent PPAR binding molecule were obtained. By using ECHLP interactive with PPAR, FLJ13111 interactive with PPAR and AOP2 interactive with PPAR, a screening system for a drug ameliorating insulin resistance is constructed and disclosed, the drug giving selectively the main action with no occurrence of the adverse action. Additionally, a method for producing a pharmaceutical composition for ameliorating insulin resistance is disclosed, which contains as the active component, a promoting agent of the main action through PPAR, an agonist specific to the main action through PPAR, an inhibitor of ECHLP interactive with PPAR to promote the main action through PPAR, a substance suppressing the adverse action through PPARγ, an inhibitor of AOP2 interactive with PPAR to suppress the adverse action through PPARγ, an activating agent of FLJ13111 interactive with PPAR to promote the main action through PPAR or an activator of FLJ13111 expression.

TECHNICAL FIELD

The present invention relates to a method for screening a proteininteractive with PPAR in a ligand-dependent manner, and a method forscreening a drug ameliorating insulin resistance, utilizing the protein.

BACKGROUND OF THE INVENTION

It has been shown that thiazolidine derivative, which is recognized tohave an effect as an insulin sensitizing agent, function as an agonistof the peroxisome proliferator activated receptor gamma (PPARγ) (seenon-patent reference 1). Since the affinity of thiazolidine derivativesfor PPARγ is in a correlation with hypoglycemic effect in human body, itis believed that the effect of this group of compound for amelioratinginsulin resistance is caused via PPARγ (see non-patent reference 2).Therefore, it has been considered that a method for detecting PPARγagonists is an effective tool to select drugs for insulin resistantdiabetes mellitus

Diabetes mellitus is caused by insufficient action of insulin secretedfrom pancreas and mainly includes two types. So-called type 1 diabetesmellitus occurs due to the damage of pancreatic β cells, so insulin isessential for the treatment. Meanwhile, type 2 diabetes mellitus(non-insulin-dependent-diabetes mellitus) occurs due to daily habitsphysically burdensome such as overeating, lack of exercise and stress inaddition to genetic factors. Type 2 diabetes mellitus occupies most ofJapanese patients with diabetes mellitus, while the number of thepatients with type 1 is very small. In patients with type 2 diabetesmellitus, insulin resistance emerges, so the promotion of glucosemetabolism via insulin hardly occurs. Therefore, research works havebeen kept on going not only about agents for simply lowering bloodglucose level as anti-diabetic agent but also about the therapeutictreatment of the subject with type 2 diabetes mellitus for promotingglucose metabolism by insulin sensitizing.

It is known that PPAR belongs to the nuclear receptor superfamily thatbinds to a response element upstream of a target gene and induces itstranscription in a ligand-dependent manner (see non-patent reference 3).

It is known that PPAR includes three subtypes, which are referred to asPPARα, PPARβ, and PPARγ (see non-patent references 4 and 5). Further,various compounds activating these PPARs, which lower blood glucose orlipid, have been reported. For example, it is known that thiazolidinederivatives as anti-diabetic agents are PPARγ ligands and significantlylower serum triglyceride level (see non-patent references 6 to 9).Alternatively, fibrate having been traditionally used as hypolipidemicagents are known to act as a ligand for PPARα. Clinically, it isobserved that the fibrate strongly lowers serum triacylglycerol level(see non-patent references 10 and 11).

It has been reported that PPARγ agonists terminate cell growth andpromote cell differentiation (see non-patent reference 12). PPARγexpression is observed particularly in fat tissues (see non-patentreferences 13 and 14), and no induction of adipocyte differentiationoccurs in PPARγ homo-deficient mice. Additionally, administration ofthiazolidine derivatives acting as PPARγ agonists induces the decreaseof large adipocytes and the increase of small adipocytes (see non-patentreference 15). Based on the findings described above, the mechanism ofthiazolidine derivatives for insulin sensitizing is believed as follows:as the consequence of the rapid promotion of adipocyte differentiationby the PPARγ agonist, the production of TNFA causing insulin resistanceis suppressed, together with the promotion of the expression of glucosetransporter in peripheral tissues and the suppression of the generationof free fatty acid; consequently, then, glucose uptake in peripheraltissues is activated and hyperglycemia is ameliorated (see non-patentreference 16).

Lately, clinical reports findings using thiazolidine derivativesindicates that all of synthetic PPARγ agonist not only have an action ofinsulin sensitizing, but also induce edema with increase of plasmavolume in vivo. (see non-patent references 17 and 18). The edema inducedby the synthetic PPARγ agonists is a severe adverse action resultingcardiac hypertrophy. Therefore, it is strongly desired that the adverseaction be separated from the main action thereof, namely theamelioration of insulin resistance. However, it has not yet beenelucidated what kind of signal pathway works for a complex of PPARγ andits ligands to induce different responses, namely the adipocytedifferentiation along with the insulin sensitizing and the induction ofedema. In other words, the molecular mechanism for these inductions hasnot yet been revealed.

Interactions with a group of transcription cofactors are necessary forthe transcriptional activation via PPARs, like other nuclear receptors.Therefore, attempts have been made so as to identify cofactorsinteracting with PPARs. Actually, biochemical approaches have been usedto examine the binding between the known cofactors for nuclear receptorsand PPARγ. It is reported that several some molecules such as SRC-1 (seenon-patent reference 19), CBP/p300 (see non-patent reference 20),DRIP205 and TRAP220 (see non-patent reference 21), SMRT (see non-patentreference 22), Gadd45 (see non-patent reference 23) and RIP140 (seenon-patent reference 24) interact with PPARγ. A report reveals thataccording to a biochemical approach, similarly, the retinoid X receptor(RXR) together with PPAR forms a heterodimer in a manner dependent onthe presence of a ligand, to bind to a response element upstream atarget gene (see non-patent reference 25). However, the detailedmechanism of the agonist dependency of these cofactors or of how thesecofactors are involved in the downstream signaling of PPAR is stillunclear.

Meanwhile, a method using the yeast two-hybrid system (see non-patentreference 26) with intervening ligands has been widely used as a methodfor screening new cofactors interactive with nuclear receptors. However,it has been difficult so far to find a ligand-dependent binding factor,in particular, for PPARγ by the yeast two-hybrid system. According tothe results of screening such PPARγ-binding factors by a yeasttwo-hybrid system with no intervening ligand, PPARγ-binding factors suchas PBP (see non-patent reference 27), PGC-1 (see non-patent reference28), PGC-2 (see non-patent reference 29), and SHP (see non-patentreference 30) have been reported. However, all the factors interact withPPARγ even in the absence of ligand. Therefore, not any apparentlyligand-dependent PPARγ binding factor could be obtained. Only a fewreports have indicated about the detection of the ligand dependency ofthe binding between PPARγ and interactive factors by the yeasttwo-hybrid system. However, the interaction was detected in thesereports using the highly concentrated cultured yeast cells expressingthe known cofactors for nuclear receptors together with PPARγ. (seepatent reference 1 and non-patent reference 24). Therefore, no successwas made in screening an apparently ligand-dependent interactive factorfor PPARγ from a cDNA library by the yeast two-hybrid system. Concerningthe aforementioned Gadd45 and PGC-1, for example, their ligand-dependentinteractions with nuclear receptors including a PPARα were detected withthe yeast two-hybrid system. However, the ligand-dependency of thesecofactors to interact with PPARγ can be observed only by the biochemicalapproach (see non-patent reference 24). It has been explained that sincethe biochemical approach and the approach using yeast differ from eachother in terms of sensitivity and the ratio of probe to interactivefactor, the action of a PPARγ ligand cannot efficiently be detected bythe yeast two-hybrid system (see non-patent reference 24). Although thebiochemical approach is suitable for detecting the interaction between apair of proteins, it is very difficult to screen for all proteinsinteractive with a certain specific protein by the biochemical approach.Meanwhile, the yeast two-hybrid system can screen proteins interactivewith an objective protein from libraries.

As described above, it has been strongly desired to separate the adverseaction of edema induction from the desirable action of amelioratinginsulin resistance, but the molecular mechanism responsible for theseparation has not yet been elucidated. Thus, it has been highly desiredthat the mechanism be elucidated, together with the development of amethod for screening a drug ameliorating insulin with a lower level ofthe adverse action.

Herein, ECHLP/Ech1 includes a structure speculated as a region for twoenzymes, namely enoyl-CoA hydratase and dienoyl-CoA isomerasefunctioning for the fatty acid metabolism within the molecule (seenon-patent reference 31). Various reports are issued about the DNAsequence of ECHLP/Ech1 (see patent references 2 to 7). However, thephysiological function is not yet elucidated. AOP2 is calledanti-oxidant protein 2 (GenBank accession No. XM_(—)001415) because AOP2includes peroxidase-like sequence within the molecule. Various reportsare issued about the DNA sequence of AOP2 (see patent reference 8 to12). As an actual physiological activity, some report indicated thatAOP2 functioned as a calcium-independent phospholipase A2 (seenon-patent reference 32), while another report indicated that the genelocus of the AOP2 was revealed as a gene causing polycystic nephropathiain mouse (see non-patent reference 33). As described above, apparently,AOP2 has an action different from the molecular function deduced on thebasis of the amino acid sequence and structure, and the originalphysiological function has not yet been identified. Although thesequence of FLJ13111 has been reported (see patent references 13 and14), a function of the protein is still unknown. There is no informationindicating the molecular function of FLJ13111 based on the amino acidsequence and structure except that the presence of a nucleus targetingsequence and the presence of a site to be possibly glycosylated withinthe molecule.

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DISCLOSURE OF THE INVENTION

By a unique approach including the presence of a high concentration of ahighly effective PPARγ agonist in the yeast two-hybrid system, thepresent inventors identified a group of proteins binding to PPARγ in amanner dependent on the presence of an agonist with a high effect oftriggering an action of ameliorating glucose metabolism (main action)and a group of proteins binding to PPARγ in a manner dependent on thepresence of an agonist with a high effect of triggering edema (adverseaction). Consequently, the inventors found ECHLP (enoyl-CoAhydratase-like protein) as a molecule binding to PPARγ in a mannerdependent on an agonist with main action and human anti-oxidant protein2 (non-selenium glutathione peroxidase; acidic calcium-independentphospholipase A2; GenBank accession No. XM_(—)001415; hereafterabbreviated as AOP2) as a molecule binding to PPARγ in a mannerdependent on an agonist with adverse action.

The inventors found that overexpression of ECHLP protein in cellssuppressed the ligand-dependent transcription-inducing activity of PPARγdistinctly. Further, the inventors found that the expression level ofECHLP gene was raised irrespective of the variation of blood glucoselevel in a diabetic model mouse compared with normal mouse by using thegene chip method. Then, the inventors confirmed that the protein was thefactor causing diabetic mellitus. The inventors additionally found thatoverexpression of AOP2 in cells promoted the ligand-dependenttranscription-inducing activity of PPARγ distinctly. Further, theinventors found the increase of AOP2 protein in the diabetic model mouseby two-dimensional electrophoresis and then confirmed that the excesspresence of the protein in diabetic mellitus activates the expression ofa specific gene group inducing edema through PPARγ.

By the unique approach including the presence of a high concentration ofa highly active PPARγ agonist in the yeast two-hybrid system similarly,the inventors found FLJ13111 (GenBank Accession No. AK023173) as amolecule binding to PPARγ in a manner dependent on the agonist withdesirable action. Further, the inventors found that overexpression ofFLJ13111 in cells activated the ligand-dependent transcription-inducingactivity of PPARγ distinctly. Additionally, the inventors confirmed thatthe expression of the FLJ13111 gene was significantly lowered in themuscle tissue of a diabetic model mouse compared with normal mouse. Theinventors first recovered the promoter region of FLJ13111 andconstructed an assay system to detect the promoter activity of FLJ13111gene. The assay system can be utilized for screening PPARγ ligands ordrugs ameliorating insulin resistance, with no use of the protein PPARγ.

Based on these findings, a new drug ameliorating insulin resistance,which makes a specific contribution to the desirable action via PPAR anddoes not induce the adverse action can be identified and additionally, amethod for screening such drug is provided.

Specifically, the present invention relates to the following:

-   (1) A method for screening a protein interactive with PPARγ in a    ligand-dependent manner, utilizing a yeast two-hybrid system in the    presence of a PPAR ligand with a high potency of triggering the    action for ameliorating glucose metabolism, wherein a polynucleotide    encoding a region containing at least the position 204 to position    505 of the PPARγ protein represented by SEQ ID NO: 2 is used as bait    and a cDNA library is used as prey.-   (2) A method for screening a protein interactive with PPARγ in a    ligand-dependent manner, utilizing a yeast two-hybrid system in the    presence of a PPAR ligand with a high potency of triggering edema,    wherein a polynucleotide encoding a region containing at least the    position 204 to position 505 of the PPARγ protein represented by SEQ    ID NO: 2 is used as bait and a cDNA library is used as prey.-   (3) A cell transformed by i) a polynucleotide encoding a polypeptide    consisting of an amino acid sequence of SEQ ID NO: 4 or a    polynucleotide encoding a polypeptide comprising an amino acid    sequence represented by SEQ ID NO: 4 wherein 1 to 10 amino acids    therein are deleted, substituted and/or inserted and also    interacting with PPAR in a ligand-dependent manner, ii) a gene    encoding a fusion protein comprising at least the ligand binding    region of the PPAR protein represented by SEQ ID NO: 2 or 6 and the    DNA binding region of a transcription factor, and iii) a reporter    gene fused to a response element to which said DNA binding region of    the transcription factor is capable of binding; or-   a cell transformed by i) a polynucleotide encoding a polypeptide    consisting of an amino acid sequence of SEQ ID NO: 4 or a    polynucleotide encoding a polypeptide comprising an amino acid    sequence represented by SEQ ID NO: 4 wherein 1 to 10 amino acids    therein are deleted, substituted and/or inserted and also    interacting with PPAR in a ligand-dependent manner and ii) a    reporter gene fused to a response element to which the DNA binding    region of the PPAR protein represented by SEQ ID NO: 2 or 6 is    capable of binding, said cell expressing a) a polypeptide consisting    of an amino acid sequence of SEQ ID NO: 4 or a polypeptide    comprising an amino acid sequence represented by SEQ ID NO: 4    wherein 1 to 10 amino acids therein are deleted, substituted and/or    inserted and interacting with PPAR in a ligand-dependent manner    and b) the PPAR protein represented by SEQ ID NO: 2 or 6.-   (4) A cell transformed by i) a polynucleotide encoding a polypeptide    consisting of an amino acid sequence of SEQ ID NO: 8 or a    polynucleotide encoding a polypeptide comprising an amino acid    sequence represented by SEQ ID NO: 8 wherein 1 to 10 amino acids    therein are deleted, substituted and/or inserted and additionally    interacting with PPAR in a ligand-dependent manner, ii) a gene    encoding a fusion protein comprising at least the ligand binding    region of the PPAR protein represented by SEQ ID NO: 2 or 6 and the    DNA binding region of a transcription factor, and iii) a reporter    gene fused to a response element to which said DNA binding region of    the transcription factor is capable of binding, or

a cell transformed by i) a polynucleotide encoding a polypeptideconsisting of an amino acid sequence of SEQ ID NO: 8 or a polynucleotideencoding a polypeptide comprising an amino acid sequence represented bySEQ ID NO: 8 wherein 1 to 10 amino acids therein are deleted,substituted and/or inserted and additionally interacting with PPAR in aligand-dependent manner and ii) a reporter gene fused to a responseelement to which the PPAR protein represented by SEQ ID NO: 2 or 6 iscapable of binding, said cell expressing a) a polypeptide consisting ofan amino acid sequence of SEQ ID NO: 8 or a polypeptide comprising anamino acid sequence represented by SEQ ID NO: 8 wherein 1 to 10 aminoacids therein are deleted, substituted and/or inserted and interactingwith PPAR in a ligand-dependent manner, and b) the PPAR proteinrepresented by SEQ ID NO: 2 or 6.

-   (5) A cell described in (3) or (4), wherein the transcription factor    is the GAL4 protein of yeast.-   (6) A cell described in (3) or (4), wherein the reporter gene is    luciferase gene.-   (7) A method for detecting whether or not a test substance promotes    the action of ameliorating glucose metabolism via PPAR,    comprising i) a step of allowing the cell described in (3), a PPAR    ligand and a test substance in contact with each other, and ii) a    step of analyzing the change of the ligand-dependent interaction or    the change of the transcriptional activity induced by    ligand-activated PPAR, using the expression of a reporter gene as a    marker.-   (8) A method for screening a drug ameliorating insulin resistance,    comprising i) a step of allowing the cell described in (3), a PPAR    ligand and a test substance in contact with each other, and ii) a    step of analyzing the change of the ligand-dependent interaction or    the change of the transcriptional activity induced by    ligand-activated PPAR, using the expression of a reporter gene as a    marker.-   (9) A method for screening as described in (8), wherein the drug    ameliorating insulin resistance is a drug ameliorating glucose    metabolism.-   (10) A method for detecting whether or not a test substance promotes    the activity triggering edema via PPAR, comprising i) a step of    allowing a test substance in contact with the cell described in (4),    and ii) a step of analyzing the change of the interaction due to the    test substance or the change of the transcriptional activity induced    via PPAR due to the test substance using the expression of a    reporter gene as a marker.-   (11) A method for screening a drug ameliorating insulin resistance    with no activity of triggering edema, comprising i) a step of    allowing a test substance in contact with the cell described in    (4), ii) a step of analyzing the change of the interaction due to    the test substance or the change of the transcriptional activity    induced via PPAR due to the test substance, using the expression of    a reporter gene as a marker; and iii) a step of selecting a test    substance not enhancing the reporter activity.-   (12) A method for screening as described in (11), wherein the drug    ameliorating insulin resistance is a drug ameliorating glucose    metabolism.-   (13) A cell transformed by i) a polynucleotide encoding a    polypeptide consisting of an amino acid sequence of SEQ ID NO: 17 or    a polynucleotide encoding a polypeptide comprising an amino acid    sequence represented by SEQ ID NO: 17 wherein 1 to 10 amino acids    therein are deleted, substituted and/or inserted and also    interacting with PPAR in a ligand-dependent manner, ii) a gene    encoding a fusion protein comprising at least the ligand binding    region of the PPAR protein represented by SEQ ID NO: 2 or 6 and the    DNA binding region of a transcription factor, and iii) a reporter    gene fused.to a response element to which said DNA binding region of    the transcription factor is capable of binding; or-   a cell transformed by i) a polynucleotide encoding a polypeptide    consisting of an amino acid sequence of SEQ ID NO: 17 or a    polynucleotide encoding a polypeptide comprising an amino acid    sequence represented by SEQ ID NO: 17 wherein 1 to 10 amino acids    therein are deleted, substituted and/or inserted and additionally    interacting with PPAR in a ligand-dependent manner and ii) a    reporter gene fused to a response element to which the PPAR protein    represented by SEQ ID NO: 2 or 6 is capable of binding, said cell    expressing a) a polypeptide consisting of an amino acid sequence of    SEQ ID NO: 17 or a polypeptide comprising an amino acid sequence    represented by SEQ ID NO: 17 wherein 1 to 10 amino acids therein are    deleted, substituted and/or inserted and interacting with PPAR in a    ligand-dependent manner, and b) the PPAR protein represented by SEQ    ID NO: 2 or 6.-   (14) A method for detecting whether or not a test substance promotes    the action of ameliorating glucose metabolism via PPAR,    comprising i) a step of allowing a test substance in contact with    the cell described in (13), and ii) a step of analyzing the change    of the interaction due to the test substance or the change of the    transcriptional activity induced via PPAR due to the test substance,    using the expression of a reporter gene as a marker.-   (15) A method for screening a drug ameliorating insulin resistance,    comprising i) a step of allowing the cell described in (13) in    contact with a test substance, and ii) a step of analyzing the    change of the interaction due to the test substance or the change of    the transcription al activity induced via PPAR due to the test    substance, using the expression of a reporter gene as a marker.-   (16) A method for screening as described in (15), wherein the drug    ameliorating insulin resistance is a drug ameliorating glucose    metabolism.-   (17) A method for screening a drug ameliorating insulin resistance,    comprising i) a step of allowing a test substance in contact with a    cell transformed with a reporter gene fused to a polynucleotide    consisting of a nucleotide sequence of SEQ ID NO: 26 or a    polynucleotide comprising a nucleotide sequence represented by SEQ    ID NO: 26 wherein 1 to 10 bases therein are deleted, substituted    and/or inserted and also having a transcription promoter activity,    and ii) a step of analyzing the change of the activity for    transcriptional induction due to the test substance, using the    expression of a reporter gene as a marker.-   (18) A method for screening as described in (17), wherein the    reporter gene is the luciferase gene.-   (19) A method for producing a pharmaceutical composition for    ameliorating insulin resistance, comprising a screening step using a    screening method described in (8), (11), (15) and/or (17) and a    formulation step using a substance obtained by the screening.

Amino acid sequences highly homologous to the full-length ECHLP of SEQID NO: 4 or a partial sequence thereof, or nucleotide sequences encodingthe amino acid sequences are reported in various papers (WO 00/55350, WO02/29103, WO 02/00677, WO 01/49716, WO 00/37643, WO 01/75067). None ofthem includes any description that ECHLP is responsible for insulinresistance. Amino acid sequences highly homologous to the full-lengthAOP2 of SEQ ID NO: 8 or a partial sequence thereof, or nucleotidesequences encoding the amino acid sequences have been reported invarious papers (WO 98/43666, Antioxid Redox Signal. 1999 Winter; 1(4):571-84. Review, WO 2002 12328, WO 2002 29086, WO 2002 06317). However,none of them includes any description that AOP2 is responsible forinsulin resistance. WO 01/55301 describes the same sequence as that ofAOP2 identified by the inventors and includes a description of the useof the sequence as a substance for regulating functions for atherapeutic treatment of diabetes mellitus among therapeutic treatmentsof various diseases. However, the publication never includes any exampleor description supporting the involvement of the sequence in diabetesmellitus. EP 1 074 617 discloses the same amino acid sequence as that ofFLJ13111 of SEQ ID NO: 17 and the nucleotide sequence encoding thesequence. However, the report never includes any description about thenames of specific diseases wherein FLJ13111 is involved. WO 00/58473discloses a sequence homologous to the nucleotide sequence of FLJ13111and includes a description of the use of the sequence as a substance forregulating functions for a therapeutic treatment of diabetes mellitusamong therapeutic treatments of various diseases. However, thepublication never includes any examples or descriptions supporting theinvolvement of the sequence in diabetes mellitus. Thus, the binding ofECHLP, AOP2 and FLJ13111 to PPAR is a finding first found by theinventors. Further, the screening of a new drug ameliorating insulinresistance by detecting a substance making a specific contributions tothe desirable action through PPAR and does not induce adverse actionsusing them is an invention first achieved by the inventors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the agonist selectivity in the binding of aligand-dependent PPARγ interactive factor and PPARγ.

FIG. 2 is a graph showing the Ech1 expression levels in diabetic modelmice KKA^(y)/Ta (KKA^(y)) and C57BL/KsJ-db/db(db/db) and normal mice forcomparison.

FIG. 3 is a graph showing the distribution of Ech1 expressed in tissues.

FIG. 4 is a graph showing the suppressive action of ECHLP on theligand-dependent transcriptional induction ability of PPARγ.

FIG. 5 is a graph showing the promoting action of AOP2 on theligand-dependent transcriptional induction ability of PPARγ.

FIG. 6 is a graph showing the screening of a PPARγ ligand specific tothe main action, utilizing the actions of ECHLP and AOP2 on theligand-dependent transcriptional induction ability of PPARγ.

FIG. 7 is a graph showing the promoting action of FLJ13111 on theligand-dependent transcriptional induction ability of PPARγ.

FIG. 8 is a graph showing the FLJ13111 expression levels in diabeticmodel mice KKA^(y)/Ta (KKA^(y)) and C57BL/KsJ-db/db(db/db) and normalmice [C57BL/6J (C57BL), C57BL/KsJ/+m(m+/m+)] for comparison.

FIG. 9 is a graph showing the transcriptional induction activity ofFLJ1311 promoter and the influence of pioglitazone or the overexpressionof FLJ13111 on the activity.

FIG. 10 is a graph showing the influence of ECHLP overexpression on theincrease of the triglyceride content via pioglitazone in murine 3T3-L1cells.

FIG. 11 is a graph showing the transcriptional induction ability ofPPARγ in the presence or absence of FLJ13111, which depends onpioglitazone or the compound XF.

FIG. 12 is a graph showing the influence of pioglitazone or the compoundXF on the expression level of sodium-potassium ATPase in renalepithelial cells.

BEST MODE FOR CARRYING OUT THE INVENTION

The terms used for the invention are now described below.

The term “main action” used in this specification represents “action forameliorating glucose metabolism”, while the term “adverse action” means“action for triggering edema”. The action for ameliorating glucosemetabolism refers to an action for promoting a function to incorporateblood sugar (glucose) into cells to consume the sugar therein andaccumulate the sugar in the form of an energy storage substance such asglycogen. The action for triggering edema refers to an effect oftriggering edema (swelling) because of the accumulation and retention ofextracellular fluids in interstitium. The term “main action ligand”means “ligand with a high potent of triggering an action forameliorating glucose metabolism (main action)” while the “adverse actionligand” means “ligand with a high potent of triggering edema (adverseaction)”. Concerning the ligand with a high potent of triggering theaction for ameliorating glucose metabolism, preferably, theconcentration of a compound requiring a 25% decrement of the bloodglucose level compared with a control group is as low as ⅕-fold or less,more preferably {fraction (1/10)}-fold or less the concentration of aPPARγ ligand of the related art (for example, pioglitazone), accordingto the blood glucose assay method of Miwa I, et. al., more preferablyunder the conditions of Example 1. The compound includes for exampleGW-7282 and GI-262570 described below. According to the blood glucoseassay method of Miwa I, et. al., blood glucose level is assayed by anenzyme method using a combination of mutarose and glucose oxidase. Theligand with a high potent of triggering edema preferably includes acompound. giving a 25% or more increment of circulating plasma volume intwo weeks compared with a control group, or giving a 15% or moreincrement of circulating plasma volume in two weeks, compared with awell-known PPARγ ligand (for example, pioglitazone), when the compoundis administered at 100 mg/kg, according to the method of Brizzee B L et.al. (J. Appl. Physiol. 69(6): 2091-2096, 1990) for assaying circulatingplasma volume, more preferably under the conditions of Example 1. Thecompound includes for example GW-7282 and GI-100085 described below.

The term “cell for testing” refers to “cell wherein the ligand-dependentinteraction between PPAR and ECHLP can be assayed using the expressionof a reporter gene as a marker”, “cell wherein the ligand-dependentinteraction between PPAR and AOP2 can be assayed using the expression ofa reporter gene as a marker”, or “cell wherein the ligand-dependentinteraction between PPAR and FLJ13111 can be assayed using theexpression of a reporter gene as a marker”. The term “yeast two-hybridsystem” means a system for detecting protein-protein interaction byutilizing the two separate function of transcription factor of yeast.The transcription factor contains the DNA binding region and thetranscription-activating region, and the interaction of both the tworegions is necessary for transcriptional activation. In the yeasttwo-hybrid system consists of two components, 1. a target protein fusedto a DNA binding region of the transcription factor and 2. a proteinfused to a transcription-activating region of the transcription factor.and the interaction of the two components can be detected by monitoringtranscriptional activation. In the yeast two-hybrid system, the baitrefers to a target protein fused to a DNA binding region, while the preyrefers to a protein fused to the transcription-activating region. The“CDNA library” is prepared by extracting and separating several tenthousands of mRNAs (copies of genetic information to instruct amino acidsequences of proteins) synthesized in cells, then synthesizingcomplimentary DNAs using the mRNAs as templates withreverse-transcription enzyme, processing the termini and integratingthen the resulting cDNAs into a vector. In this specification, “PPARligand-binding region” refers to a region where a ligand of PPAR binds,individually including the region including the position 204 to position505 of the amino acid sequence of SEQ ID NO: 2 for human PPARγ2 and theregion including the position 167 to position 468 of the amino acidsequence of human PPARα. The “DNA binding region” is a regionfunctioning for DNA binding and has a DNA binding ability to a responseelement but has no transcription-activating ability of its own. The DNAbinding region of the GAL4 transcription factor exists on the N-terminalside (the region comprising amino acids at about position 1 to aboutposition 147).

The present invention is now described in detail hereinbelow.

In this specification, the PPAR-interactive polypeptide encoded by thepolynucleotide contained in the gene of a protein interactive with PPARfor preparing a cell for testing includes

-   (1) a polypeptide consisting of an amino acid of SEQ ID NO: 4, 8 or    17;-   (2) a polypeptide comprising an amino acid sequence represented by    SEQ ID NO: 4, 8 or 17 wherein 1 to 10 amino acids therein are    deleted, substituted and/or inserted and which is a protein binding    to PPAR in a ligand-dependent manner (referred to as functionally    equivalent variant hereinafter); and-   (3) a polypeptide consisting of an amino acid sequence with 90% or    more homology to the amino acid sequence of SEQ ID NO: 4, 8 or 17,    which is a protein binding to PPAR in a ligand-dependent manner    (referred to as homologous peptide hereinafter).

The functionally equivalent variant is preferably “a polypeptidecomprising an amino acid of SEQ ID NO: 4, 8 or 17, which is a proteinbinding to PPAR in a ligand-dependent manner”, “a polypeptide comprisingan amino acid sequence represented by SEQ ID NO: 4 or 17 wherein 1 to10, preferably 1 to 7, more preferably 1 to 5 amino acids therein aredeleted, substituted and/or inserted and which is a protein binding toPPAR in a manner depending on the main action ligand”; or “a polypeptidecomprising an amino acid sequence represented by SEQ ID NO: 8 wherein 1to 10, preferably 1 to 7, more preferably 1 to 5 amino acids therein aredeleted, substituted and/or inserted and which is a protein binding toPPAR in a manner depending on the adverse action ligand.

The homologous polypeptide consisting of an amino acid sequence with 90%or more homology to the amino acid sequence of SEQ ID NO: 4, 8 or 17 andis a protein binding to PPAR in a ligand-dependent manner, with nospecific limitation. The homologous polypeptide consisting of an aminoacid sequence with preferably 90% or more, more preferably 95% or more,still more preferably 98% or more homology to the amino acid sequence ofSEQ ID NO: 4 or 17 and is a protein binding to PPAR, preferably in amanner depending on the main action ligand. The homologous polypeptideconsisting of an amino acid sequence with preferably 90% or more, morepreferably 95% or more, still more preferably 98% or more homology tothe amino acid sequence of SEQ ID NO: 8 and is a protein binding toPPAR, preferably in a manner depending on the adverse action ligand. Inthis specification, additionally, the ‘homology’ refers to the valueobtained using parameters preset as default by the Clustal program(Higgins and Sharp, Gene 73, 237-244, 1998; Thompson et al., NucleicAcid Res. 22, 4673-4680, 1994). The parameters are as follows.

Pairwise alignment parameters are as follows.

-   K tuple 1-   Gap Penalty 3-   Window 5-   Diagonals Saved 5.

The PPAR-interactive polypeptides contained in the cell for testing inthis specification are described above. The polypeptide consisting of anamino acid of SEQ ID NO: 4, functionally equivalent variants thereof andhomologous polypeptides thereof are collectively referred to as “ECHLPinteractive with PPAR” hereinbelow. The polypeptide consisting of anamino acid of SEQ ID NO: 8, functionally equivalent variants thereof andhomologous polypeptides thereof are collectively referred to as “AOP2interactive with PPAR” hereinbelow. The polypeptide consisting of anamino acid of SEQ ID NO: 17, functionally equivalent variants thereofand homologous polypeptides thereof are collectively referred to as“FLJ13111 interactive with PPAR” hereinbelow.

The polynucleotide of a nucleotide sequence encoding the ECHLPinteractive with PPAR, the AOP2 interactive with PPAR or the FLJ13111interactive with PPAR may be any of the polynucleotide encoding theamino acid sequence of SEQ ID NO: 4, 8 or 17, a functionally equivalentvariant thereof or a polynucleotide comprising a nucleotide sequenceencoding a homologous polypeptide thereof. Preferably, thepolynucleotide is a polynucleotide consisting of a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 4, 8 or 17. Morepreferably, the polynucleotide consisting of a nucleotide sequence ofSEQ ID NO: 3, 7 or 16.

A method for screening a protein interacting with PPAR in aligand-dependent manner, which works as a useful tool for screening adrug ameliorating insulin resistance without the adverse action is nowdescribed below, together with a method for screening a drugameliorating insulin resistance without the adverse action, utilizingthe protein.

[A Method for Screening a Protein Interacting with PPAR in aLigand-Depending Manner]

In accordance with the present invention, all the protein factorsinteracting with PPARγ in a ligand-dependent manner can be identifiedfrom cDNA libraries using as a marker the expression of a reporter geneof the yeast two-hybrid system. In accordance with the presentinvention, the ligand-dependent interaction between PPAR and atranscription cofactor thereof is detected with no need of the detectionof the transcriptional induction ability of PPAR per se. Accordingly,factor groups inherent to mammals are not necessary, which are involvedin the expression of the transcription induction ability of PPAR.Therefore, mammalian cells are not necessarily used specifically as thecell for testing. Therefore, eukaryotic cells, for example yeast cells,insect cells and mammalian cells are also satisfactory. Among them,yeast cells can readily be cultured in a rapid way. Additionally,genetic recombination techniques such as the introduction of exogenousgenes are readily applicable to the cells. Still additionally, theligand dependency of the binding between PPAR and the interactivefactors can be followed up and detected efficiently by a method usingthe same yeast two-hybrid system.

The yeast two-hybrid system is a method for detecting a protein-proteininteraction using as a marker the expression of a reporter gene.Generally, a transcription factor includes functionally different tworegions, namely a DNA binding region and a transcription activatingregion. In order to examine the interaction between two proteins types Xand Y by the two-hybrid system, two protein types namely a fusionprotein comprising the DNA binding region of a transcription factor andX and a fusion protein comprising the transcription activating region ofa transcription factor and Y are simultaneously expressed in yeastcells. When the proteins X and Y interact with each other, the two typesof the fusion proteins form one transcription complex, which binds to aresponse element (a DNA site for specific binding) of the transcriptionfactor in the cell nucleus to activate the transcription of the reportergene arranged downstream the response element. As described above, theinteraction of the two proteins can be detected as the detection of theexpression of the reporter gene.

The yeast two-hybrid system is generally used for identifying an unknownprotein interacting with a specific protein, using the specific proteinas probe. When the binding of the two occurs in a manner depending onthe presence of a receptor ligand as observed in the case of a nuclearreceptor and a group of some of the transcription coupling factorsthereof, a two-hybrid system with a ligand added extraneously to thesystem should be used. As described above in the Section “Background ofthe Invention”, however, it was difficult to detect the liganddependency between PPARγ and the interactive factors by the yeasttwo-hybrid system. Thus, no success was made in screening for all theligand-dependent PPARγ interactive factors. The inventors assumed thatthe reason might be that the PPARγ agonists might have low intracellularpermeability because of the yeast properties so that the detectionsensitivity of the ligand dependency would be low. By reacting acompound group with the highest activity as the PPARγ agonist amongthose reported with yeast, the inventors achieved a unique method forthe yeast two-hybrid system applicable to the assay of the liganddependency between PPARγ and the interactive factors and to thescreening. More specifically, the screening can be carried out by amethod described in Example 2.

Another embodiment of the method including detecting a ligand-dependentinteractive factor with PPARγ to assay the action of a test substance onthe interaction is a method for biochemically detecting theligand-dependent binding between PPARγ and the interactive factor. Bysuch a method, a protein binding to a fusion protein comprising anappropriate tag protein such as glutathione-S-transferase (GST), proteinA, β-galactosidase, and maltose-binding protein (MBP) and the ligandbinding region of PPARγ is directly detected in the presence of a testsubstance in an extract solution of a culture cell labeled with forexample RI; then, the binding protein is purified and determined of itsamino acid sequence, for identification.

[Method for Detecting an Action Ameliorating Glucose Metabolism andMethod for Screening a Drug Ameliorating Insulin Resistance, Utilizing aProtein Interacting with PPAR in a Ligand-Dependent Manner; Method forDetecting an Aactivity Trigger Edema and Method for Screening a DrugAmeliorating Insulin Resistance with no Activity Triggering Edema,Utilizing a Protein Interacting with PPAR in a Ligand-Dependent Manner]

1. Method for Detecting an Action Ameliorating Glucose Metabolism andMethod for Screening a Drug Ameliorating Insulin Resistance, UtilizingECHLP Interactive with PPAR

One Embodiment of the present invention is a method for detectingwhether or not a test substance can selectively promote the main actionthrough PPAR, using a cell for testing, which is preliminarilytransformed with (i) a fusion gene of at least the ligand binding regionof PPARα or γ and the DNA binding region of a transcription factor orthe gene encoding the full-length PPARα or γ molecule, (ii) the geneencoding the ECHLP interactive with PPAR, and (iii) a reporter geneconjugated to a response element to which the DNA binding region of thetranscription factor is capable of binding or a reporter gene conjugatedto a response element to which PPARα or γ is capable of binding,comprising a step of making the cell for testing concurrently presentwith the test substance in the presence of a PPAR ligand, detecting andassaying the change of the suppressive action of the ECHLP interactivewith PPAR on the transcription activating ability of PPAR due to thetest substance in the cell for testing as the expression of the reportergene as a marker. An additional embodiment is a method for screening acompound selectively promoting the main action via PPAR by selecting acompound enhancing the reporter activity as detected by the detectionmethod.

2. Method for Detecting the Activity Triggering Edema and Method forScreening a Drug Ameliorating Insulin Resistance with no ActivityTriggering Edema, Utilizing AOP2 Interactive with PPAR

One embodiment of the present invention is a method for detecting acompound with the adverse action via PPAR, using a cell for testing,which is preliminarily transformed with (i) a fusion gene of at leastthe ligand binding region of PPARα or γ and the DNA binding region of atranscription factor or the gene encoding the full-length PPARα or γmolecule, (ii) the gene encoding the AOP2 interactive with PPAR, and(iii) a reporter gene conjugated to a response element to which the DNAbinding region of the transcription factor is capable of binding or areporter gene conjugated to a response element to which PPARα or γ iscapable of binding, comprising a step of making the cell for testingconcurrently present with a test substance, detecting and assaying thechange of the promoting action of the AOP2 interactive with PPAR on thetranscription activating ability of PPAR due to the test substance inthe cell for testing as the expression of the reporter gene as a marker,together with a method for selecting and screening a compoundselectively promoting the desirable action without the adverse actionwith the reporter system.

3. Method for Detecting an Action Ameliorating Glucose Metabolism andMethod for Screening a Drug Ameliorating Insulin Resistance, UtilizingFLJ13111 Interactive with PPAR

One embodiment of the present invention is a method for detectingwhether or not a test substance can selectively promote the desirableaction through PPAR, using a cell for testing, which is preliminarilytransformed with (i) a fusion gene of at least the ligand binding regionof PPARγ and the DNA binding region of a transcription factor or thegene encoding the full-length PPARγ molecule, (ii) the gene encoding theFLJ13111 interactive with PPAR, and (iii) a reporter gene conjugated toa response element to which the DNA binding region of the transcriptionfactor is capable of binding or a reporter gene conjugated to a responseelement to which PPARα or γ is capable of binding, comprising a step ofmaking the cell for testing concurrently present with the testsubstance, detecting and assaying the change of the promoting action ofthe FLJ13111 interactive with PPAR on the transcription activatingability of PPAR due to the test substance in the cell for testing as theexpression of the reporter gene as a marker. An additional embodiment isa method for screening a compound selectively promoting the desirableaction via PPAR by selecting a compound enhancing the reporter activityas detected by the detection method.

In the embodiment 1, 2 or 3 above, the transcription factor to be usedfor the detection of the transcriptional induction ability of PPARincludes but is not limited to any eukaryotic transcription factors witha region binding to a specific DNA sequence in cell nucleus.Additionally, the DNA binding region of such transcription factor has aDNA binding ability to a response element but does not have atranscription activating ability of its own. Such transcription factorincludes for example yeast GAL4 protein (Keegan, et al., Science,Vol.231, p. 699-704, 1986, Ma, et al., Cell, Vol. 48, p. 847-853, 1987).In case of GAL4, for example, the DNA binding region and transcriptionactivating region of the GAL4 transcription factor exist on the aminoterminus(a region containing amino acids, approximately at position 1 toposition 147).

As the response element, a DNA sequence to which the DNA binding regionof a transcription factor is capable of binding is used. The region iscut out from the upstream region of the gene or the region may bechemically prepared synthetically for use.

The reporter gene to be arranged downstream the response elementincludes but is not specifically limited to any reporter gene forgeneral use. As such, enzyme genes quantitatively assayable readily arepreferable. The reporter gene includes for example chloramphenicolacetyltransferase gene (CAT), firefly-derived luciferase gene (Luc), andgreen fluorescence protein gene (GFP) from jellyfish. The reporter geneis functionally conjugated to the downstream of the response element.

A polynucleotide encoding PPARα or γ, the DNA binding region of atranscription factor, the ECHLP interactive with PPAR, AOP2 interactivewith PPAR, or FLJ13111 interactive with PPAR can be isolated from cDNAlibraries, by the polymerase chain reaction (PCR) or hybridization,using primers and probes designed and synthetically prepared on thebasis of the information of known amino acid sequences and nucleotidesequences. The ECHLP interactive with PPAR may be derived from anyspecies as long as the resulting ECHLP can be identified as the samemolecular species and interacts with PPAR in a ligand-dependent mannerto influence the transcription induction ability of the receptor. TheECHLP interactive with PPAR includes those from mammalian animals, forexample humans (LOC115289; GenBank Accession No. XM_(—)008904, HPXEL;GenBank Accession No. U16660, FitzPatrick DR, et al., Genomics 1995Vol.27 (3): p. 457-466), mouse (Ech1; GenBank Accession No.NM_(—)016772), and rat (HPXEL; GenBank accession No. NM_(—)022594,FitzPatrick DR, et al., Genomics 1995 Vol.27 (3): p.457-466).

The AOP2 interactive with PPAR may be derived from any species as longas the resulting AOP2 can be identified as the same molecular speciesand interacts with PPAR in a ligand-dependent manner to influence thetranscription induction ability of the receptor. The AOP2 interactivewith PPAR includes those from mammalian animals for example humans(AOP2/KIAA0106; GenBank Accession No. XM_(—)001415, D14662), mouse(AOP2/1-Cys Prx/nonselenium glutathione peroxidase; GenBank AccessionNo. AF004670, AF093852, Y12883), rat (AOX2; GenBank Accession No.AF014009), and cow (GPX/PHGPx; GenBank Accession No. AF080228,AF090194).

The FLJ13111 interactive with PPAR may be derived from any species aslong as the resulting FLJ13111 can be identified as the same molecularspecies and interacts with PPAR in a ligand-dependent manner toinfluence the transcription induction ability of the receptor. TheFLJ13111 interactive with PPAR includes those from mammalian animals forexample humans (FLJ13111; GenBank Accession No. AK023173, NM_(—)025082)and mouse (human FLJ13111-like protein; GenBank Accession No.XM_(—)134598).

PPARγ may be derived from any species as long as the resulting PPARγ canbe identified as the same molecular species and can function as anuclear receptor in biological organisms. PPARγ includes for examplethose derived from mammalian animals such as human, mouse and rat andfrom Xenopus. The gene sequence and amino acid sequence of PPARγ havebeen reported (Dryer, et al., Cell, Vol. 68, p.879-887, 1992, Zhu, etal., Journal of Biological Chemistry, Vol. 268, p. 26817-26820, 1993,Kliewer, et al., Proc. Natl. Acad. Sci. USA, Vol. 91, p. 7355-7359,1994, Mukherjee, et al., Journal of Biological Chemistry, Vol. 272, p.8071-8076, 1997, Elbrecht, et al., Biochem. Biophys. Res. Commun.,Vol.224, p. 431-437, 1996, Chem, et al., Biochem. Biophys. Res. Commun.,Vol. 196, p. 671-677, 1993, Tontonoz, et al., Genes & Development, Vol.8, p.1224-1234, 1994, Aperlo, et al., Gene, Vol. 162, p. 297-302, 1995).Additionally, PPARγ includes two isoform types, namely PPARγ1 andPPARγ2. Compared with PPARγ2, PPARγ1 is deficient in the 30 amino acidson the amino terminus thereof. The remaining amino acid sequence istotally the same. It is known that both of them are expressed in fattissues.

A polynucleotide encoding PPARα or γ, the DNA binding region of atranscription factor, the ECHLP interactive with PPAR, AOP2 interactivewith PPAR or FLJ13111 interactive with PPAR can be obtained for examplein the following way. With no limitation to the method, thepolynucleotide can be obtained by the known procedure described in“Molecular Cloning”, “Sambrook, J., et al., Cold Spring HarborLaboratory Press, 1989”.

mRNA comprising one encoding the protein can be extracted by a knownmethod from cells or tissues with an ability to generate the protein,for example a fat tissue with the ability. The extraction methodincludes for example the guanidine/thiocyanate/hot phenol method and theguanidine/thiocyanate-guanidine/hydrochloric acid method, preferablyguanidine/thiocyanate cesium chloride method. A cell or tissue with anability to generate PPARα or γ, ECHLP interactive with PPAR, AOP2interactive with PPAR or FLJ13111 interactive with PPAR can beidentified by Northern blotting using a gene comprising the nucleotidesequence encoding the protein or a part thereof, Western blotting usingan antibody specific to the protein, and the like.

mRNA can be purified by conventional methods. For example, mRNA isadsorbed onto oligo (dT) cellulose column, which can then be eluted forpurification. Further, mRNA can be fractionated further by sucrosedensity gradient centrifugation method. Additionally, commerciallyavailable mRNA extracted may satisfactorily be used, without the mRNAextraction procedure.

Then, the purified mRNA is applied to a reverse-transcription enzymereaction in the presence of random primer or oligo dT primer, tosynthetically prepare a first cDNA chain. The synthesis can be done byconventional methods. Using the resulting first cDNA chain and twoprimer types directed for a partial region of the intended gene, forexample SEQ ID NOS: 9 and 10 for PPARγ, SEQ ID NOs: 12 and 13 for theECHLP interactive with PPAR, SEQ ID NOs: 14 and 15 for the AOP2interactive with PPAR, or SEQ ID NOs: 18 and 19 for the FLJ13111interactive with PPAR, the CDNA is treated by PCR, to amplify thesequence of the intended gene. Using a commercially available cDNAlibrary, additionally, similar two primer types directed for a partialregion of the intended gene may be used for PCR, to amplify the sequenceof the intended gene. The resulting DNA is fractionated by agarose gelelectrophoresis and the like. If desired, the DNA is digested withrestriction enzymes. By subsequently conjugating the resulting products,an intended DNA fragment can be obtained. Specifically, such intendedDNA fragment can be obtained by the methods described in Examples 2, 4,5, 7, 8, 10 and 11.

The determination of the sequence of the DNA obtained by the methodsdescribed above can be done by the chemical modification method of Maxamand Gilbert (Maxam, A. M. and Gilbert, W., “Methods in Enzymology”, 65,499-559, 1980) or the dideoxynucleotide chain termination method(Messing, J. and Vieira, J., Gene, 19, 269-276, 1982) or the like.

By the method described in “Molecular Cloning”, “Sambrook, J., et al.,Cold Spring Harbor Laboratory Press, 1989”, DNAs encoding theseindividual regions are used singly or are conjugated together, forconjugation to the downstream of an appropriate promoter, to constructan expression system of PPARα or γ and the ECHLP interactive with PPARin cells in vitro, and an expression system of PPARα or γ and the AOP2interactive with PPAR in cells in vitro. In the same manner, anexpression system of PPARγ and the FLJ13111 interactive with PPAR incells in vitro can be constructed.

Specifically, the polynucleotide thus obtained may be integrated in anappropriate vector plasmid and then inserted in the plasmid form into ahost cell. These may satisfactorily be constructed so that the two maybe contained in one plasmid or the two may be contained separately inindividually different plasmids. Otherwise, a cell with suchconstruction integrated in the chromosomal DNA may satisfactorily beobtained and then used.

As to the reporter gene conjugated to a response element, the reportergene is constructed using general gene recombination techniques; theresulting construct is once integrated in a vector plasmid; then, theresulting recombinant plasmid is inserted into a host cell; and theresulting reporter gene inserted in such manner is used. Otherwise, suchconstruct is integrated into the chromosomal DNA of a cell, and theresulting cell is obtained to use the construct as it is.

PPAR may satisfactorily be inserted extraneously. In case that afat-derived cell or a kidney-derived cell abundant in endogenous PPARγis used as a host cell, a construct consisting of only a reporterconjugated to a response element and the ECHLP interactive with PPARexcluding PPARγ, a construct consisting of only a reporter conjugated toa response element and the AOP2 interactive with PPAR excluding PPARγ, aconstruct consisting of only a reporter conjugated to a response elementand the FLJ13111 interactive with PPAR excluding PPARγ maysatisfactorily be inserted.

More specifically, a fragment containing the isolated polynucleotide isagain integrated in an appropriate vector plasmid, to thereby transforman eukaryotic or prokaryotic host cell. By further inserting anappropriate promoter and a sequence involved in gene expression intosuch vector, the gene can be expressed in the resulting individual hostcells.

For example, the eukaryotic host cell includes cells of vertebraeanimals, insects and yeast. As the cells of vertebrae animals, thefollowing ones are often used: a monkey cell COS cell (Gluzman, Y.(1981) Cell, 23, 175-182), dihydrofolate-deficient Chinese hamster ovarycell (CHO) (Urlaub, G. and Chasin, L. A. (1980) Proc. Natl. Acad. Sci.USA, 77, 4216-4220), human embryonic kidney-derived HEK293 cell and293-EBNA cell (manufactured by Invitrogen) prepared by inserting theEBNA-1 gene of Epstein Barr virus into the cell mentioned above.However, the cell is not limited to those described above. Any cell maybe satisfactory, wherein the inhibition of the transcription inductionability of PPARα or γ with the ECHLP interactive with PPAR or thetranscription induction activity of PPARα or γ with the AOP interactivewith PPAR or the transcription induction activity of PPARγ with theFLJ13111 interactive with PPAR can be detected.

As the expression vector of vertebrae cells, generally, an expressionvector with a promoter, RNA splicing sites, polyadenylation sites, atranscription termination sequence and the like as located upstream agene to be expressed may satisfactorily be used. If necessary, theexpression vector may have an origin of replication. The expressionvector includes for example but is not limited to pSV2dhfr with theearly SV40 promoter (Subramani, S. et al., (1981) Mol. Cell. Biol., 1,854-864), pEF-BOS with a human elongation factor promoter (Mizushima, S.and Nagata, S. (1990) Nucleic acids Res., 18, 5322), and pCEP4 with acytomegalovirus promoter (manufactured by Invitrogen).

A case of using the COS cell as the host cell is exemplified now. Anexpression vector with an origin of SV40 replication and autonomousproliferation ability in the COS cell and additionally with atranscription promoter, a transcription termination signal and an RNAsplicing site may be used and includes pME18S (Maruyama, K. and Takebe,Y. (1990) Med. Immunol., 20, 27-32), pEF-BOS (Mizushima, S. and Nagata,S. (1990) Nucleic Acids Res., 18, 5322), and pCDM8 (Seed, B. (1987)Nature, 329, 840-842). The expression vector can be incorporated in theCOS cell, by the DEAE-dextran method (Luthman, H. and Magnusson, G.(1983) Nucleic Acids Res., 11, 1295-1308), the calcium phosphate-DNAcoprecipitation method (Graham, F. L. and van der Ed, A. J. (1973)Virology, 52, 456-457), the method by means of FuGENE6 (manufactured byBoehringer Mannheim), and electroporation with electric pulse (Neumann,E. et al. (1982) EMBO J., 1, 841-845). In such manner, a desiredtransformant cell can be obtained.

In case of using the CHO cell as such host cell, a vector capable ofexpressing the neo gene functioning as a G418 resistant marker, forexample pRSVneo (Sambrook, J. et al. (1989): “Molecular Cloning-ALaboratory Manual”, Cold Spring Harbor Laboratory, NY) and pSV2-neo(Southern, P. J. and Berg, P. (1982) J. Mol. Appl. Genet., 1, 327-341)is cotransfected together with the expression vector. By selecting aG418-resistance colony, a transformant cell stably generating theprotein group can be obtained. In case of using the 293-EBNA cell as thehost cell, additionally, an expression vector such as pCEP4 (Invitrogen)with an origin of Epstein Barr virus replication and autonomousproliferation ability in the 293-EBNA cell is used to obtain a desiredtransformant cell.

The resulting transformant obtained above can be cultured byconventional methods. Through the culturing, the intended protein groupis generated in the cell. As the culture medium for use in theculturing, various culture media routinely used for the host cellselected can be selected appropriately. For the COS cells for example,culture media such as the RPMI-1641 culture medium and the Dulbecco'smodified Eagle's minimum essential culture medium (DMEM) supplementedfor example with the serum component of fetal bovine serum (FBS) on aneeded basis may be used. For the 293-EBNA cells, additionally, theDulbecco's modified Eagle's minimum essential culture medium (DMEM)supplemented for example with the serum component of fetal bovine serum(FBS) and additionally supplemented with G418 can be used.

Culturing a cell for testing in the presence of a test substance, theinhibition of the suppressive action of the ECHLP interactive with PPARon the transcription induction ability of PPARα or γ due to the testsubstance can be detected and assayed on the basis of the expression ofthe reporter gene. (1) When a test substance reacts with the ECHLPinteractive with PPAR or with PPAR and the suppressive effect of theECHLP interactive with PPAR on the transcription induction activity ofPPAR is reduced in a manner dependent on the action, it is observed thatthe reporter activity expressed reaches maximum. Such test substance canbe identified as a promoting agent of the main action through PPAR.Additionally (2) when a test substance binds to PPAR to promote thetranscription induction ability while the test substance inhibits thesuppressive effect of the ECHLP interactive with PPAR, the increase ofthe expressed reporter activity is observed. Such test substance isidentified as an agonist specific to the main action through PPAR.Further (3) when a test substance binds to the ECHLP interactive withPPAR to inhibit the suppressive effect of the transcription inductionability of PPAR, or when a test substance inhibits the expression of theECHLP interactive with PPAR or promotes the decomposition thereof, it isobserved similarly that the expressed reporter activity increases. Suchsubstance can be identified as an inhibitor of the ECHLP interactingwith PPAR to promote the main action through PPAR. Expectantly, any ofthese (1), (2) and (3) acts as a drug ameliorating insulin resistance,without the adverse action brought about by a PPAR agonist. Morespecifically, a drug ameliorating insulin resistance can be identifiedand screened by the methods described in Examples 5 and 9. Under theconditions described in Example 9, for example, a substance with IC50 of10 μM or less, preferably 1 μM or less can be selected as a drugameliorating insulin resistance.

Culturing a cell for testing in the presence of a test substance, it canbe detected and assayed on the basis of the expression of the reportergene that the promoting action of the AOP2 interactive with PPAR on thetranscription induction ability of PPARα or γ can be suppressed by thetest substance. (1) When a test substance reacts with the AOP2interactive with PPAR or with PPARγ to reduce the promoting effect ofthe AOP2 interactive with PPAR on the transcription induction ability ofPPARγ in a manner depending on the action, the decrease of the reporteractivity expressed is observed. Such test substance can be identified asa substance suppressing the adverse action through PPARγ. Additionally(2) when a test substance binds to PPARγ to promote the transcriptioninduction ability while the test substance inhibits the promoting effectof the AOP2 interactive with PPAR, it is observed that the reporteractivity expressed is decreased to the same level as the state with noconcurrent expression of the AOP2 interactive with PPAR. Such testsubstance is identified as an agonist selective to the main actionthrough PPARγ without the adverse action. Further (3) when a testsubstance binds to the AOP2 interactive with PPAR to inhibit thepromoting effect of the transcription induction ability of PPARγ, orwhen a test substance inhibits the expression of the AOP2 interactivewith PPAR or promotes the decomposition thereof, it is observedsimilarly that the expressed reporter activity decreases. Such substancecan be identified as an inhibitor of the AOP2 interactive with PPAR tosuppress the adverse action through PPARγ. Expectantly, any of them actsas a drug ameliorating insulin resistance without the adverse actionbrought about by a PPARγ agonist. Meanwhile, when a test substancereacts for example with the AOP2 interactive with PPAR or with PPARγ toactivate the promoting effect of AOP2 interactive with PPARγ on thetranscription induction activity of PPARγ in a manner depending on theaction, the increase of the reporter activity expressed is observed.Such test substance is identified as a substance strongly triggering theadverse action through PPARγ, so a drug ameliorating insulin resistancewith no activity inducing edema can be screened by selecting a testsubstance never involving the increase of the reporter activity.

Culturing a cell for testing in the presence of a test substance, theactivation with a test substance of the promoting action of the FLJ13111interactive with PPAR on the transcription induction ability can bedetected and assayed on the basis of the expression of the reportergene. (1) When a test substance acts with the FLJ13111 interactive withPPAR or with PPARγ to enhance the promoting effect of the FLJ13111interactive with PPAR on the PPARγ transcription induction activity in amanner dependent on the action, it is observed that the reporteractivity expressed increases. Such test substance can be identified as apromoting agent of the main action through PPARγ. Additionally (2) whena test substance binds to PPAR to promote the transcription inductionability while the test substance enhances the promoting effect of theFLJ13111 interactive with PPAR, such test substance is identified as anagonist specific to the main action through PPAR. Further (3) when atest substance binds to the FLJ13111 interactive with PPAR to enhancethe promotion effect of the transcription induction ability of PPAR orwhen a test substance promotes the expression of the FLJ13111interactive with PPAR or suppresses the decomposition thereof, it isalso observed that the reporter activity expressed is increasedsimilarly. Such substance is identified as an activating agent of theFLJ13111 interactive with PPAR, to promote the main action through PPAR.Expectantly, any of these (1), (2) and (3) acts as a drug amelioratinginsulin resistance without the adverse action brought about by a PPARagonist. More specifically, a drug ameliorating insulin resistance canbe identified and screened by the methods described in Examples 11 and12. Under the conditions described in Example 12, for example, asubstance with ED50 of 10 μM or less, preferably 1 μM or less can beselected as a drug ameliorating insulin resistance.

[Method for Screening a Drug Ameliorating Insulin Resistance Utilizing aPromoter for FLJ13111 Interactive with PPAR]

-   i) A drug ameliorating insulin resistance can be screened by a    method comprising i) a step of allowing a test substance in contact    with a cell transformed with a reporter gene fused to a    polynucleotide consisting of the nucleotide sequence of SEQ ID NO:    26 or a polynucleotide comprising the nucleotide sequence of SEQ ID    NO: 26 wherein 1 to 10 amino acids therein are deleted, substituted    and/or inserted and also having a transcription promoter activity    and ii) a step of analyzing the change of the transcription    activity-inducing activity with the test substance, using as a    marker the expression of the reporter gene.

The reporter gene assay (Tamura, et al., Transcription Factor ResearchMethod, Yodosha, 1993) is a method for assaying the regulation of geneexpression using as the marker the expression of a reporter gene.Generally, gene expression is regulated with a part called promoterregion existing in the 5′-upstream region thereof. The gene expressionlevel at the stage of transcription can be estimated by assaying theactivity of the promoter. When a test substance activates a promoter,the transcription of the reporter gene arranged downstream the promoterregion is activated. In such manner, the expression of the reporter genecan be detected in place of the promoter-activating action, namely theaction of activating the expression. Thus, the expression of thereporter gene can be detected in place of the action of a test substanceon the regulation of the expression of the FLJ13111 interactive withPPAR, by the reporter gene assay using the promoter region of theFLJ13111 interactive with PPAR. As the “reporter gene” to be fused tothe FLJ13111 promoter region consisting of the nucleotide sequence ofSEQ ID NO: 26, any reporter gene for general use is satisfactory with nospecific limitation. For example, an enzyme gene readily assayablequantitatively is preferable. For example, the reporter gene includeschloramphenicol acetyltransferase gene (CAT) derived from bacteriatranspozon, luciferase gene (Luc) derived from firefly and greenfluorescence protein gene (GFP) derived from jellyfish. The reportergene may satisfactorily be fused functionally to the FLJ13111 promoterregion consisting of the nucleotide sequence of SEQ ID NO: 26. Bycomparing between the expression level of the reporter gene in case thata test substance is in contact with a cell transformed with the reportergene fused to the promoter region of the FLJ13111 interactive with PPARand the expression level thereof in case that a test substance is not incontact with the reporter gene, the change of the transcriptioninduction activity depending on the test substance can be analyzed. Bycarrying out the step, screening a substance activating the expressionof FLJ13111 and a substance ameliorating insulin resistance can be done.Specifically, the screening can be carried out by the method describedin Example 14.

The test substance for use in the screening method of the inventionincludes but is not limited to commercially available compounds(including peptides), various known compounds (including peptides)registered in the chemical files, a group of compounds obtained by thecombinatorial chemistry technique (N. K. Terrett, M. Gardner, D.W.Gordon, R. J.Kobylecki, J. Steele, Tetrahedron, 51, 8135-73 (1995)),bacterial culture supernatants, naturally occurring components derivedfrom plants and marine organisms, animal tissue extracts or compounds(including peptides) chemically or biologically modified from compounds(including peptides) selected by the screening method of the invention.

[Method for Producing a Pharmaceutical Composition for AmelioratingInsulin Resistance]

The present invention encompasses a method for producing apharmaceutical composition for ameliorating insulin resistance,comprising a screening step using the screening method of the inventionand a formulation step using a substance obtained by the screening.

The formulation containing the substance obtained by the screeningmethod of the invention as the active component can be prepared, usingcarriers, excipients and/or other additives for general use in theformulation of the active component, depending on the type of the activecomponent.

The dosing includes oral dosing via tablets, pills, capsules, granules,fine granules, powders or oral liquids, or parenteral dosing viaintravenous and intramuscular injections or injections into joints,suppositories, transcutaneous dosage forms or transmembrane dosageforms. For peptides to be digested in stomach, in particular, parenteraldosing such as intravenous injection is preferable.

A solid composition for oral dosing contains one or more activesubstances and at least one inert diluents, such as lactose, mannitol,glucose, microcrystalline cellulose, hydroxypropyl cellulose, starch,polyvinylpyrrolidone or magnesium aluminate metasilicate. Thecomposition may contain additives other than inert diluents, for examplelubricants, disintegrators, stabilizers or dissolution agents orauxiliary dissolution agents according to general methods. If necessary,tablets or pills may be coated with films such as sugar coating orstomach-soluble or enteric coatings.

The oral liquid composition may include for example emulsions,solutions, suspensions, syrups or elixirs and may contain inert diluentsfor general use, for example distilled water or ethanol. The compositionmay contain additives other than inert diluents, for example,emollients, suspending agents, sweeteners, flavoring agents orpreservatives.

Non-parenteral injections may include aseptic, aqueous or non-aqueoussolutions, suspensions or emulsions. The aqueous solutions orsuspensions may contain for example water for injection or physiologicalsaline as diluents. The diluents for non-aqueous solutions orsuspensions include for example propylene glycol, polyethylene glycol,plant oils (for example, olive oil) and alcohols (for example, ethanol),or polysorbate 80. The composition may contain an emollient, anemulsifying agent, a dispersant, a stabilizer, a dissolution agent or anauxiliary dissolution agent, or a preservative. The composition can besterilized by filtration through bacteria-trapping filters, blending ofsterilizing agents or irradiation. Additionally, an aseptic solidcomposition is produced, which is then dissolved in aseptic water orother aseptic media for injection prior to use and is then used.

The dose can be appropriately determined, in view of the activecomponent, namely a substance inhibiting the activation of the LTRPC2protein or the intensity of the activity of a substance obtained by thescreening method of the invention, the symptom, and age or sex of asubject for its dosing.

In case of oral dosing, for example, the daily dose is about 0.1 to 100mg, preferably 0.1 to 50 mg per adult (with a body weight of 60 kg). Incase of parenteral dosing in the form of an injection, the daily dose is0.01 to 50 mg, preferably 0.01 to 10 mg.

EXAMPLES

The invention is now described in detail in the following Examples.However, the Examples never limit the present invention. Unlessotherwise stated, herein, the invention may be carried out according tothe known method (“Molecular Cloning”, Sambrook, J., et al., Cold SpringHarbor Laboratory Press, 1989, etc.). In case of using commerciallyavailable reagents or kits, the invention is also carried out accordingto the instructions of the commercially available products.

(Example 1) Identification of Main Action Ligand and Adverse ActionLigand

Five types of thiazolidine derivatives reported to act as PPARγ agonistswere the following compounds: GW7282[(S)-3-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl]-2-(1-pyrolyl)propionicacid; Glaxo Smith Kline, Drug Data Rep 2001, 23(9): 889], Gl-262570[(S)-2-[(2-benzoylphenyl)amino]-3-[4-[2-(5-methyl-2-phenyloxazol-4-yl)ethoxy]phenyl]propionicacid; Glaxo Smith Kline, WO 00/38811],GL-100085[2-(3-(2-(5-methyl-2-phenyloxazol-4-yl)ethoxy)phenylmethylthio)aceticacid; Ono Pharmaceutical Co., Ltd, WO 99/46232], rosiglitazone[(±)-5-[4-[2-[N-methyl-N-(2-pyridyl)amino]ethoxylbenzyl]-2,4-thiazolidinedionemaleate; Glaxo Smith Kline, WO 01/47529], pioglitazone[(+)-5-[4-[2-(5-ethyl-2-pyridinyl)ethoxy]benzyl]-2,4-thiazolidinedione;Takeda Chemical Industries, Ltd., JP-A-61-267580]. So as to elucidatethe action mechanisms thereof, the compounds were first syntheticallyprepared according to the methods reported in the patent specificationsor references thereof. In the presence of these compounds, the mainaction and the adverse action were individually measured by usinganimals. The resulting effects were quantified. Herein, the action oflowering blood glucose as a marker of the main action and the increaseof the circulating plasma volume as a marker of the action of triggeringedema were measured (Masa-aki Arakawa, Latest Internal Medicine Outline(Saishin Naikagaku Taikei), Vol. 3, Main Symptoms; Diagnosis based onSymptoms, 260-266, 1966; Kanazawa, et al., Diabetes Frontier, Vol.10, p.811-818, 1999; Iwamoto, Diabetes Frontier, Vol.10, p.819-824, 1999).

(1) Assaying the Action for Lowering Blood Glucose in the CompoundAdministrated Group

The individual compounds suspended in 0.5% methyl cellulose (MC) andsubsequently adjusted to a final concentration (1-10 mg/kg) were orallygiven once daily to KKA^(y)/Ta mice of age 7 to 8 weeks (Clea Japan,Inc.) continuously for 4 days. Only 0.5% MC was given to a controlgroup. 16 hours after the final dosing, blood was taken out from themurine tail vein. The blood glucose level was assayed with a commercialkit (Glucose CII Test Wako, Wako Pure Chemical Industries, Ltd.) usingan enzyme method by means of a combination of mutarose and glucoseoxidase (see Miwa I, et al., Clin Chim Acta Vol.37, p. 538, 1972).Provided that the blood glucose level in the control group was definedas 100%, the compound concentration (ED25) estimated to reduce the bloodglucose level in the control group by 25% was calculated by linearregression using the least square method (Table 1).

(2) Assaying the Activity for Triggering Edema of the Compound Group

A test compound was orally dosed once daily at a dose of 100 mg/kg(suspended in 0.5% methyl cellulose) to rats (Sprague-Dawley rats; malesof age 3 weeks) continuously for 2 weeks. The plasma volume was assayed,fundamentally according to the method described in J. Appl. Physiol.69(6): 2091-2096, 1990. 0.25% Evans Blue solution (in physiologicalsaline) was administrated by intravenous injection into the lower leg ofthe rats at 0.25 ml(0.625 mg)/rat under anesthesia with ether. Fiveminutes later, blood was taken out from the inferior vena cava. Theplasma was diluted with water. The Evans Blue concentration (mg/ml)based on the absorbance (620 nm) was divided by the injection amount(0.625 mg). The resulting value was defined as plasma volume. Further,the ratio (%) of the plasma volume on a body weight basis to that in thecontrol group (vehicle-dosed group) was calculated (Table 1)

As the results of them, GW7282 strongly triggered the main action andthe adverse action. Meanwhile, GI-262570 was at a relatively high valueof triggering the main action but showed a weak action. Additionally,GL-100085 poorly triggered the main action but strongly triggered theadverse action. TABLE 1 Blood glucose-lowering action and circulatingplasma volume-increasing action of PPARγ agonists Hypoglycemic TestCirculating plasma ED25 (mg/kg) % of CTRL GW-7282 0.41 130 GI-2625700.98 124 GL-100085 17 133 Pioglitazone 10 110 Rosiglitazone 4.6 114

(Example 2) Identification of Protein Interacting with PPARγ in aLigand-Dependent Manner

(1) Isolation of PPARγ Gene

cDNA encoding the C-terminal 302 amino acids including the DNA bindingregion and ligand binding region of PPARγ was obtained from a cDNAlibrary derived from human fat tissues (Clontech: Marathon Ready™ cDNA)by polymerase chain reaction (PCR) . In order to insert the cDNA into anexpression vector pDBtrp (Invitrogen; containing TRP1 gene as aselective marker) for the yeast two-hybrid on the basis of the genesequence of human PPARγ2 as described as the GenBank Accession No.U79012 in the gene database, regions homologous to the 40 nucleotidesbefore and after the multicloning site of the vector was added to theCDNA. Further, primers of SEQ ID NOs.: 9 and 10 were designed so thatindividual recognition sites for restriction enzymes KpnI and SmaI wereadded on both the ends of the inserted gene fragment of PPARγ. Using aDNA polymerase (Pyrobest DNA polymerase; manufactured by TaKaRa, Co.,Ltd.), PCR was done at 98° C. (for one minute) and subsequently byrepeating a cycle of 98° C. (5 seconds)/55° C. (30 seconds) and 72° C.(3 minutes) 35 times. Consequently, the resulting DNA fragment of 1004base pairs (bp) includes the coding region of PPARγ, which consists of302 amino acids from the 204-th amino acid of the PPARγ2 to the aminoacid immediately before the termination codon.

(2) Preparation of Expression Plasmid for use in Yeast Two-Hybrid

The vector pDBtrp linearized by digestion with restriction enzymes SalIand NcoI and the PCR fragment containing the cDNA of PPARγ as obtainedin (1) were simultaneously added to a yeast strain MaV203 (Invitrogen)for use in the two-hybrid, for transfection by the lithium acetatemethod (C Guthrie, R Fink Guide to Yeast Genetics and Molecular Biology,Academic, San Diego, 1991). Consequently, homologous recombinationoccurred in the yeast cell, so that a plasmid with the PPARγ cDNAinserted at the multicloning site of pDBtrp (abbreviated as pDB-PPARγhereinbelow) was formed. The yeast cell carrying the plasmid wasselected by culturing the cell on the solid synthetic minimum essentialculture medium (DIFCO) (20% agarose) deficient in tryptophan as aselective marker of the plasmid. The yeast cell was treated withZymolyase (Seikagaku Corporation) at ambient temperature for 30 minutes.Subsequently, the plasmid was isolated and purified by the alkali method(“Molecular Cloning”, Sambrook, J., et al., Cold Spring HarborLaboratory Press, 1989), for the determination of the nucleotidesequence using a sequencing kit (Applied BioSystems) and a sequencer(ABI 3700 DNA sequencer, Applied BioSystems). Thus, a plasmid with theinserted cDNA of PPARγ was selected, where the reading frame of the cDNAof PPARγ matches with the reading frame of the pDBtrp region encodingthe DNA binding region of GAL4.

(3) Yeast Two-Hybrid Screening

The yeast strain MaV203 for use in the two-hybrid as transformed withthe pDB-PPARγ was suspended in 400 ml of the YPD culture broth (DIFCO)and cultured under agitation at 30° C. for about 6 hours until theabsorbance at a wavelength of 590 nanometer reached 0.1 to 0.4.Subsequently, the resulting cell was prepared into a competent cell bythe lithium acetate method. The final volume was suspended in 1.0 ml of0.1 M lithium-Tris buffer. The cell was transformed with 20 μg each ofthe human kidney cDNA library, human liver library or human skeletalmuscle (all from Clontech; Match Maker cDNA library). The resultingcells were screened by culturing on a solid synthetic minimum essentialculture medium (DIFCO) (20% agarose) deficient in tryptophan and leucineas selective markers for the pDB-PPARγ plasmid and the library plasmid,respectively, to obtain a transformant strain with both the plasmidsinserted therein. Concurrently, the transformant cell was cultured at30° C. for 5 days on the solid minimum essential culture medium fromwhich histidine other than tryptophan and leucine was preliminarilyeliminated and which was preliminarily supplemented with 20 mM 3AT(3-AMINO-1,2,4-TRIAZOLE; Sigma) as an inhibitor of the enzyme encoded bya reporter gene HIS3, so as to select a cell expressed in case that afused protein of the GAL4 transcription-promoting region bound to anartificially expressed fusion protein of the GALA DNA binding region inthe two-hybrid system, where the reporter gene HIS3 was involved. APPARγ agonist GW7282 strongly triggering both the main action and theadverse action was preliminarily added to the culture medium to a finalconcentration of 1.5 μM, to obtain a 3AT-resistant yeast colony showingthe expression of a protein binding to PPARγ in the presence of theagonist. These yeast cells were grown on the YPD solid culture medium ata state with addition of the agonist GW7282 at a concentration of 15 μMor with no such addition for 24 hours. Subsequently, the expression of abinding-indicating reporter for the two-hybrid system, as a differentreporter from HIS3, namely the lacZ gene, was examined, using as amarker the β-galactosidase activity. The β-galactosidase activity wasassayed by transferring the yeast cells on the culture medium onto anitrocellulose filter, freezing then the cells in liquid nitrogen,thawing the resulting cells at ambient temperature, leaving the filterto stand alone on a filter immersed with 0.4% X-GAL (Sigma) solution at37° C. for 24 hours and measuring the change of blue color withβ-galactosidase. By selecting a colony with the cell contentstransferred onto the filter being changed from white to blue, pluralyeast cells expressing a protein binding to PPARγ in a manner dependingon the presence of the agonist were identified. According to the methoddescribed in the Yeast Protocols Handbook of Clontech, a plasmid derivedfrom the library was extracted from the cells. The nucleotide sequenceof the gene fragment contained therein was sequenced, using thenucleotide sequence of SEQ ID NO: 11 (the sequence binding to the GAL4ADregion; derived from Cloning vector pACT2 under GenBank Accession No.U29899) as primer and a sequencing kit (Applied BioSystems) and asequencer (ABI 3700 DNA sequencer by Applied BioSystems). Consequently,it was verified by the homology screening by BLAST (NCBI) that clonescontaining a partial sequence of SEQ ID NO: 3 from ECHLP were containedin any of those derived from the three types of libraries. Additionally,clones containing a gene fragment of SRC-1 (Smith C L, et al., Natl.Acad. Sci. USA, Vol.20, No. 93(17), p. 8884-8888, 1996) and N-CoR (NagyL., et al., Cell, Vol. 89, No.3, p.373-380 (1997)) were contained inthose derived from the kidney-derived library. Thus, it was confirmedthat the ligand-dependent coupling factor of PPARγ could be obtained bythe screening described above.

Additionally, the same yeast two-hybrid screening was performed underthe following conditions. As a library, a cell transformed with thehuman kidney cDNA library was used. GW7282 was added to a finalconcentration of 1 μM. A yeast cell expressing a protein binding toPPARγ in the presence of the agonist was grown in the YPD solid culturemedium for 24 hours at a state with GW7282 added to a concentration of10 μM. By assaying the β-galactosidase activity, plural yeast cellsexpressing the protein binding to PPARγ in a manner depending on thepresence of the agonist were identified. From the cells, a plasmidderived from the library was extracted. The nucleotide sequence of thegene fragment contained therein was sequenced. Consequently, twoindependent clones containing a partial sequence of SEQ ID NO: 7 fromAOP2 (GenBank Accession No.: XM_(—)00415) were contained therein.Additionally, a clone containing gene fragments of SRC-1 (Smith C L, etal., Pro. Natl. Acad. Sci. USA, Vol.20, No. 93(17), p. 8884-8888, 1996)and N-CoR (Nagy L., et al., Cell, Vol. 89, No.3, p.373-380 (1997)) knownas transcriptional cofactors of nuclear receptors were containedtherein. It was now verified that the ligand-dependent transcriptionfactor of PPARγ could be obtained by the screening.

Additionally, the same yeast two-hybrid screening was performed underthe following conditions. As a library, cells transformed with the humanliver cDNA library were used. GW7282 was added to a final concentrationof 1 μM. A yeast cell expressing a protein binding to PPARγ in thepresence of the agonist was grown in the YPD solid culture medium for 24hours at a state with GW7282 added to a concentration of 10 μM. Byassaying the β-galactosidase activity, plural yeast cells expressing theprotein binding to PPARγ in a manner depending on the presence of theagonist were identified. From the cells, a plasmid derived from thelibrary was extracted. The nucleotide sequence of the gene fragmentcontained therein was sequenced. Consequently, a clone containing apartial sequence of the novel gene of SEQ ID NO: 16 (FLJ13111-analogousgene; a one-base substituted gene from GenBank Accession No.: AK023173)was contained.

(Example 3) Detection of Ligand-Selective Interaction Between PPARγ andECHLP or AOP2

The agonist dependency of the interaction between a protein group mainlyincluding ECHLP and AOP 2 as obtained in Example 2 and PPARγ wasassayed, using two types of agonists with different effects on the mainaction and the adverse action, namely GI-262570 (at a finalconcentration of 5 μW or 0.5 μM) and GL-100085 (at a final concentrationof 5 μM or 0.5 μM) and the β-galactosidase activity in the yeasttwo-hybrid system as a marker (FIG. 1;solid arrow and striped arrowpoint out larger changes of the interactions due to the difference inconcentration between the main action-selective compound and the adverseaction-selective compound, respectively; open arrow points out largerchanges of the interactions due to the difference in concentrationbetween the main action- and adverse action-selective compounds). Thedetails of the method are the same as in Example 2 except for theagonists used. Consequently, the compound with a higher effect on themain action, namely GI-262570 induced the binding between PPARγ andECHLP similarly even when the concentration was lowered from 5 μM to 0.5μM (FIG. 1 b), while the compound with a relatively high effect on theadverse action, namely GL-100085 profoundly reduced the binding betweenPPARγ and ECHLP when the concentration was lowered from 5 μM to 0.5 μM(FIG. 1 c). Alternatively, the compound GL-100085 with a relatively higheffect on the adverse action similarly induces the binding between PPARγand AOP2 even when the concentration was lowered from 5 μM to 0.5 μM(FIG. 1 c), while the compound GI-262570 with a high effect on theaction when added significantly reduced the binding between PPARγ andAOP2 even when the concentration was lowered from 5 μM to 0.5 μM (FIG. 1b). These may be due to the possible occurrence of a ligand-dependentinteraction between PPARγ and ECHLP or between PPARγ and AOP2 because ofthe presence of the agonists GI-262570 and GL-100085. The resultsapparently indicate that ECHLP-interacts with PPARγ at a highsensitivity due to the agonist with a high effect on the main action.Alternatively, it is shown that AOP2 interacts with PPARγ at a highsensitivity due to the agonist with a high effect on the adverse action(FIG. 1 c). The results indicate the presence of coupling factorsinteractive with PPARγ in an agonist-dependent manner in correlationwith the main action of an agonist or the adverse action of an agonist.ECHLP makes a more selective response to the agonist causing a strongerexpression of the main action to interact with PPARγ. It was consideredthat by utilizing the ligand-dependent interaction between PPARγ andECHLP, an agonist with a higher effect on the main action could bedetected selectively. Meanwhile, the clones #1, 4, 5, 6, 7 and N-CoRshowed lower binding levels with PPARγ when the concentration of any ofthe agonists GI-262570 and GL-100085 was reduced, so that no correlationwith the main action of the agonists or with the adverse action thereofwas observed in the clones.

(Example 4) Assaying ECHLP Expression Level in Normal Mice and DiabeticModel Mice

Based on the findings described above, it was anticipated that theinteraction between ECHLP and PPARγ might be involved in theamelioration of glucose metabolism as the main action via PPARγ agonist.Therefore, the expression level of the messenger RNA (mRNA) of the mouseortholog ech1 gene in the ECHLP gene was assayed in skeletal muscle andfat in two diabetic model types of mice, namely KKA^(y)/Ta (Iwatsuka, etal., Endocrinol. Japan., Vol.17, p. 23-25, 1970, Taketomi, et al., Horm.Metab. Res., Vol.7, p. 242-246, 1975) and C57BL/KsJ-db/db (Chen, et al.,Cell, Vol. 84, p. 491-495, 1996, Lee, et al., Nature, Vol. 379, p.632-635, 1996, Kaku, et al., Diabetologia, Vol. 32, p. 636-643, 1989),using DNA arrays (Affimetrix) (de Saizieu, et al., Nature Biotechnology,Vol. 16, p.45-48, 1998, Wodicka, et al., Nature Biotechnology, Vol. 15,p. 1359-1367, 1997, Lockhard, et al., Nature Biotechnology, Vol.14, p.1675-1680, 1996), to compare the results with those in normal individualmice C57BL/6J and C57BL/KsJ-+m/+m.

(1) Resection of Mouse Tissues:

Male eight mice of each of C57BL/6J, KKA^(y)/Ta, C57BL/KsJ-+m/+m andC57BL/KsJ-db/db were purchased from Clea Japan, Inc. The C57BL/6J micewere fed as a group with general diet until 15 weeks old. The KKA^(y)/Tamice were fed singly with a high calories diet (CMF, Oriental Yeast Co.,Ltd.) until 15 weeks old. The C57BL/KsJ-+m/+m mice and theC57BL/KsJ-db/db mice were fed as groups with general diet until 12 weeksold. It was confirmed that compared with the normal mice, the KKA^(y)/Taand C57BL/KsJ-db/db mice were hyperglycemic at larger body weights(KKA^(y)/Ta mice: blood glucose level at 514.2±18.2 mg/dl, body weightat 49.9±0.7 g; C57BL/KsJ-db/db mice: blood glucose level at 423.7±14.1mg/dl, body weight at 48.6±0.5 g). The blood glucose level was assayedby taking blood from murine caudal vein and using a commerciallyavailable kit by means of the glucose oxidase method (Autopack A/glucosereagent, Boehringer Mannheim). These four murine species wereanesthetized under diethyl ether, for resecting epididymal fat tissuesand gastrocnemius muscles. Immediately after the resection, thesetissues were frozen in liquid nitrogen and stored at −80° C.

(2) mRNA Extraction:

The tissues were disrupted using a cryo-press disruption apparatusCRYO-PRESS CP-100 (Microtec Nition). Adding ISOGEN as an RNA extractionreagent (Nippon Gene), the resulting mixture was homogenized using ahomogenizer ULTRA-TURRAX T-8 (IKA Labortechnik) . According to themanufacturer's instructions, RNA was extracted from these samples. Theresulting RNA was treated with DNase (Nippon Gene), to decompose thecontaminating DNA. Subsequently, the RNA was prepared by thephenol/chloroform extraction and ethanol precipitation, and was thendissolved in RNase-free H₂O. Using an RNA preparation reagent QuickPrepMicro mRNA Purification kit (Amersham) and according to themanufacturer's instructions, mRNA was extracted.

(3) Preparation of Labeled cDNA:

According to the instructions of Affimetrix (GeneChip ExpressionAnalysis Technical Manual), a first strand cDNA, a second strand cDNAand biotin-labeled cRNA were synthetically prepared from mRNA, and then,the labeled cRNA was fragmented.

(4) Hybridization:

The DNA array of Affimetrix (GeneChip U74) consists of 3 subarray sheetsA, B and C. According to the instructions of Affimetrix, the labeledcRNA was hybridized with the DNA array and was then rinsed, for assayingthe fluorescent intensity of each probe.

(5) Correction of Inter-Array Assay Values:

The assay values were corrected in an inter-sample manner andsubsequently in an inter-array manner. The inter-sample correction wasdone by first determining the total value of the fluorescent intensitiesof genes on a specific subarray in a inter-sample manner and thenmultiplying the assay value of each gene on another subarray by acertain magnification factor per subarray so that the total valuethereof might be equal to the array with the largest total value of thefluorescent intensities. Correction in an inter-subarray manner was doneby determining the mean value of the fluorescent intensities of AFFXprobe of each subarray and multiplying the assay value of each gene by acertain magnification factor per subarray so that the mean valuesthereof might be equal to each other among the subarrays A, B and C.

Consequently, it was confirmed that KKA^(y)/Ta mice of age 15 weeksapparently with the onset of the disease showed the expression level ofech1 mRNA 2-fold or more, compared with the 5-week-old KKA^(y)/Ta micewith no progress in the onset of the disease or the normal mice (FIG.2). Similarly, the expression level of ech1 in the db/db mice wasincreased 2-fold or more compared with that in the normal mice.

Phlorizin is known as a resorption inhibitor of the glucose deliverythrough the uriniferous tubule in kidney. When phlorizin wasadministered at a dose of 100 mg/kg at an interval of 30 minutes threetimes in the abdominal cavity of KKA^(y)/Ta mice of age 15 weeks and theblood glucose level of the mice was back to the normal level, theactivation of the ech1 expression level in the KKA^(y)/Ta mice of age 15weeks did not change in the tissues 7 hours after the firstadministration of phlorizin. Therefore, it was considered that theexpression of ech1 was not activated due to the change of the bloodglucose level as the consequence of diabetic symptoms but the activationof the expression was one of the causative factors triggering diabetesmellitus.

Using the same DNA array as described above, the mRNA expression levelfor ech1 was assayed in each organ of a male normal mouse C57BL/6J ofage 12 weeks. Consequently, the expression of ech1 was prominent in fat,muscle, liver and kidney involving the PPARγ action, and also in heartand lung (FIG. 3). In view of the expression sites, additionally, thissupports that ECHLP/Ech1 is the coupling factor of PPARγ.

(Example 5) Detection of ECHLP Regulatory Action on the Ligand-DependentTranscription Induction Ability of PPARγ

The results described above indicated that ECHLP interacted with PPARγthrough the ligands and was thereby involved in the main action(amelioration of glucose metabolism) and that the activated expressionhad some relation with symptoms of diabetes mellitus. Therefore, whatkind of influences ECHLP had on the transcription induction activity ofPPARγ was examined by reporter assay using a culture cell COS-1.

(1) Preparation of Plasmid GAL-PPARγ for Expression in Animal Cells

A chimera protein-encoding gene with cDNA encoding the ligand bindingregion of human PPARγ2 being fused to the C terminus of the DNA bindingregion (1-147 amino acids) of yeast Gal4 was integrated in themulticloning site in an animal cell expression vector pZeoSV(Invitrogen), to prepare an expression plasmid GAL-PPARγ. First, cuttingout a DNA fragment encoding the DNA binding region of Gal4 from theplasmid pGBT9 (Clontech), using restriction enzymes HindIII and SmaI,the resulting DNA fragment was inserted at the site of the multicloningsite of pZeoSV (abbreviated as pZeo-DB hereinafter). Then, cutting out aDNA fragment encoding the ligand binding region of PPARγ from theplasmid pDB-PPARγ, using KpnI and SmaI, the resulting DNA fragment wasinserted in between KpnI and PvuII sites located in the multicloningsite of pZeo-DB, to prepare an animal cell expression plasmid GAL-PPARγ.

(2) Preparation of a Plasmid pcDNA-ECHLP for Expression in Animal Cells

Using the primers of SEQ ID NOS: 12 and 13, cDNA fragment comprising 987bp (base pairs) encoding the full-length ECHLP was obtained from thehuman skeletal muscle CDNA library (Clontech) by PCR. PCR was done at98° C. (1 minute) and subsequently by carrying out a cycle of 98° C. (5seconds)/55° C. (30 seconds) and 72° C. (3 minutes) repeatedly 35 times.The resulting cDNA fragment was inserted in pcDNA3.1/V5-HIS-TOPO vector(Invitrogen) by in vitro recombination by the TOPO cloning method(Invitrogen), to prepare a plasmid pcDNA-ECHLP for expression in animalcells. Herein, no termination codon was inserted for the ECHLP.Additionally, a primer was designed so that the vector-derived V5epitope and HIS6 tag might be fused at the C terminus.

(3) Detection of ECHLP Regulatory Action on the Ligand-DependentTranscription Induction Ability of PPARγ

The culture cell COS-1 was cultured to a 70-% confluent state in aculture dish of 6-well culture plate (35-mm well diameter), to which 2ml of the minimum essential culture medium DMEM (Gibco) supplementedwith 10% fetal bovine serum (Sigma) was preliminarily added to eachwell. By the calcium phosphate method (Graham L., et al., Virology, Vol.52, p. 456, 1973, Naoko Arai, Gene Introduction andExpression/Analytical Method, p. 13-15, 1994), the cell was transientlycotransfected with the GAL-PPARγ (0.15 μg/well) and a reporter constructwith the GAL4 binding region being repeatedly arranged in a number ofeight upstream the luciferase gene (RE×8-Luci; Shimokawa, et al.,International Publication No. WO 99/04815) (0.8 μg/well), together withpcDNA-ECHLP (0.05-0.2 μg/well). After 2 μM of the PPARγ agonist or atest compound was added to the culture medium for 48-hour culturing, theculture medium was discarded and the cells were rinsed with a phosphatebuffered saline (abbreviated as PBS hereinafter). Subsequently, a celllysis solution (100 mM calcium phosphate, pH 7.8, 0.2% Triton X-100) wasadded at 0.4 ml per each well, to make the cell lytic. To 100 μl of thesolution of the lytic cell was added 100 μl of a luciferase substratesolution (Picker Gene), to assay the emission of light for 10 secondsusing a chemiluminescence assay apparatus of Type AB-2100 (ATTO).Plasmid pCH110 (Amersham Pharmacia Biotech) with the luciferase reportergene and concurrently with the β-galactosidase expression gene wascotransfected at 0.4 μg/well into the cell, to assay the β-galactosidaseactivity using a detection kit of β-galactosidase activity, namelyGalacto-Light Plus™ system (Applied BioSystems) to express the activityin numerical figure. As the numerical figure was defined as thetransfection efficiency of the introduced gene, the luciferase activitywas corrected per each well.

As the results of the experiment, it was observed that the transcriptioninduction activity of PPARγ in an agonist-dependent manner wasdistinctly inhibited, in a manner depending on the dose of the ECHLPexpression plasmid transfected into the cell (FIG. 4). This apparentlyindicates that the occurrence of the ligand-dependent interactionbetween PPARγ and ECHLP suppressed the transcription induction activityof PPARγ. This fact highly coincides with the result showing that excessECHLP/Ech1 was a causative factor of the disease in the diabetic modelmice. In other words, it was considered that the occurrence of excessexpression of ECHLP/Ech1 in the diabetic symptoms suppressed thetranscription induction activity of PPARγ, so that insufficientexpression of the downstream gene to be induced by PPARγ inhibitedglucose metabolism.

ECHLP/Ech1 includes therein a structure speculated as a region foractivating two enzyme types, namely enoyl-CoA hydratase and dienoyl-CoAisomerase working for fatty acid metabolism within the molecule(Filppula A, et al., Biol. Chem., Vol. 273, No. 1: p. 349-355, 1988).Additionally, it has been known previously that inhibitors of enzymesfor fatty acid metabolism reduce blood glucose levels in diabetic mice(Collier R, et al., Horm. Metab. Res., Vol.25, No. 1: p.9-12, 1998).Based on the fact and the aforementioned finding that ECHLP has anaction of suppressing the PPARγ activity, the presence of excess ECHLPsuppresses glucose metabolism via PPARγ to promote the energy generationfrom lipid with the own enzyme activity for fatty acid metabolism. WhenECHLP is reduced, ECHLP releases the PPARγ activity to shift thebiological energy sources toward glucose metabolism. Thus, ECHLP wasconsidered as a molecule responsible for the antagonistic regulation ofglucose and fat metabolisms. When the amount of ECHLP interactive withPPARγ is reduced or when the suppressive action of PPARγ with theinteractive ECHLP is inhibited, using ECHLP, biological energy sourcesmay possibly be directed toward glucose metabolism to reduce bloodglucose level. Using ECHLP, simultaneously, a compound with such actioncan readily be selected.

(Example 6) Comparison of the Amount of AOP2 Protein in Normal andDiabetic Model Mice

Based on the finding, it was deduced that the interaction between AOP2and PPARγ might be involved in the triggering of edema as an adverseaction via a PPARγ agonist. Therefore, the contents of proteins in fatsof diabetic model mouse KKA^(y)/Ta (Iwatsuka, et al., Endocrinol.Japon., Vol. 17, p. 23-35, 1970, Taketomi, et al., Horm. Metab. Res.,Vol.7, p. 242-246, 16975) and normal mouse C57BL/6J were compared toeach other, using the fluorescence-labeled two-dimensional differenceelectrophoresis (Unlu, et al., Electrophoresis, Vol. 18, p. 2071-2077,1997, Tonge, et al., Proteomics, Vol. 1, p. 377-396, 2001). A group ofproteins of which the contents differed by two-fold or more in thediabetic model mouse were analyzed by mass spectrometry, so as toidentify the individual proteins.

(1) Resection of Murine Tissues

Male C57BL/6J and KKA^(y)/Ta mice were purchased from Clea Japan, Inc.The C57BL/6J mice were fed in a group with general diet until 12 weeksold. The KKA^(y)/Ta mice were singly fed with a high calories diet (CMF,Oriental Yeast Co., Ltd.) until 12 weeks old. Compared with the normalmice, it was confirmed that the KKA^(y)/Ta mice were at higher bloodglucose levels and with larger body weights (KKA^(y)/Ta mice: bloodglucose value of 514.2±18.2 mg/dl; body weight of 49.9±0.7 g).Subsequently, the two types of mice were anesthetized with diethylether, to resect the fat of epididymis. Immediately after resection, theepididymis was frozen in liquid nitrogen and stored at −80° C.

(2) Preparation of Protein Samples

The frozen fat of epididymis was homogenized in a Tris buffer containingurea and an ampholytic detergent using a homogenizer ULTRA-TURRAX T-8(Ika Labortechnik). According to the protocol attached by themanufacturer, centrifugation was done to obtain the supernatants ofthese samples for use as samples for two-dimensional electrophoresis.

(3) Two-Dimensional Electrophoresis

The protocol of Amersham Pharmacia Biotech was followed. By measuringthe absorbance of each of the samples, the amount of the proteinscontained therein was determined. Taking out an amount of a samplecontaining about 50 μg of proteins, the protein was labeled withindividually different fluorescent dyes (Cy3 and Cy5, Amersham PharmaciaBiotech), and the labeled proteins were mixed together forfirst-dimensional isoelectric electrophoresis using an IPG strip(Amersham Pharmacia Biotech). Prior to second-dimensionalelectrophoresis, the IPG strip was equilibrated with a Tris buffercontaining urea, sodium dodecylsulfate, glycerol, and dithiothreitol andfurther equilibrated with a Tris buffer containing urea dissolvingiodo-acetamide therein, sodium dodecylsulfate, glycerol, anddithiothreitol. The second-dimensional electrophoresis was done usingsodium dodecylsulfate polyacrylamide electrophoresis. The gel aftercompletion of the second-dimensional electrophoresis was applied to afluorescent imaging apparatus (Amersham Pharmacia Biotech) at excitationand detection wavelengths specific to the individual fluorescent dyes,to obtain the individual two-dimensional electrophoretic images. Thesetwo images were quantified using an analytical software (AmershamPharmacia Biotech), to identify a spot with a difference in proteincontent by two-fold or more and cut out the spot with a spot pickingapparatus (Amersham Pharmacia Biotech). Then, the protein was fragmentedby the in-gel digestion method using trypsin (Schevchenko, et al.,Analytical Chemistry, Vol. 68, p. 850-858, 1996), to recover a peptidemixture from the gel.

(4) Protein Identification by Mass Spectrometry

The peptides in the resulting peptide mixture were separated by anacetonitrile gradient elution method on a capillary reverse-phase liquidchromatography column (0.075-mm diameter, 150-mm length, LC Packing) ata flow rate preset to about 200 nL per minute in the presence of 0.2%formic acid. By a quadrupole ion trap mass spectrographic apparatus(Thermoquest) with an electrospray ion source directly connected to aliquid chromatography apparatus (Microme Bioresource), automatically,the product ion spectrum of each peptide was obtained by a methodcomprising a step of selecting the molecular ion of each peptide andmeasuring the product ion spectrum.

Individual product ion spectra of fragment peptides of a peptide in thefat of the epididymis of the KKA^(y)/Ta mice, as certified of theincrease of the 2-fold or more content compared with the normal mice,were examined and compared with an analytical software Mascot (MatricScience), using a public protein database MSDB (Release 20010401).Consequently, the protein matched at partial amino acid sequences at itsfour positions with the murine AOP2 protein (AOP2/1-Cys Prx/non-seleniumglutathione peroxidase; GenBank accession No.: AF004670, AF093852,Y12883). Thus, it was revealed that the protein was the murine AOP2protein. Accordingly, this apparently indicates that the content of theAOP2 protein increases in diabetes mellitus.

(Example 7) Comparison of AOP2 Expression Levels in Tissues

Using the primers of SEQ ID NOS: 14 and 15, a 673-bp (base pairs) cDNAfragment encoding AOP2 as derived from the human cDNA library (Clontech)was amplified by PCR [using DNA polymerase (Pyrobest DNA polymerase;Takara Shuzo, Co., Ltd.) at 98° C. (1 minute) and subsequently byrepeating a cycle of 98° C. (5 seconds)/55° C. (30 seconds) and 72° C.(3 minutes) 35 times], which was then detected by agarose gelelectrophoresis. Consequently, the expression of AOP2 was distinct infat, muscle, liver and kidney with PPARγ actions among the main organs,in addition to heart. This supports even based on the expressed sitesthat AOP2 is a transcriptional cofactor of PPARγ.

(Example 8) Detection of AOP2 Regulatory Action on the Ligand-DependentTranscription Induction Ability of PPARγ

The results described above indicate that AOP2 interacts with PPARγ viaa ligand to be involved in the triggering of edema and that theactivation of the expression has a relation with diabetic symptoms.Therefore, a reporter assay using a culture cell COS-1 was done toexamine what kind of influences AOP2 had on the transcription inductionactivity of PPARγ.

(1) Preparation of Plasmid pcDNA-AOP2 for Expression in Animal Cells

Using the primers of SEQ ID NOS: 14 and 15, a cDNA fragment comprising673 bp (base pairs) encoding the full-length AOP2 was obtained from thehuman kidney cDNA library (Clontech) by PCR [using DNA polymerase(Pyrobest DNA polymerase; Takara Shuzo, Co., Ltd.) at 98° C. (1 minute)and subsequently by repeating a cycle of 98° C. (5 seconds)/55° C. (30seconds) and 72° C. (3 minutes) 35 times]. This was inserted inpCDNA3.1/V5-His-TOPO vector (Invitrogen) by in vitro recombination bythe TOPO cloning method (Invitrogen), to prepare a plasmid pcDNA-AOP2for expression in animal cells. Herein, no termination codon wasinserted for the AOP2. Additionally, a primer was designed so that thevector-derived V5 epitope and HIS6 tag might be fused at the C terminus.

(2) Detection of AOP2 Regulatory Action on the Ligand-DependentTranscription Induction Ability of PPARγ

The culture cell COS-1 was cultured to a 70-% confluent state in aculture dish of 6-well culture plate (35-mm well diameter), to which 2ml of the minimum essential culture medium DMEM (Gibco) supplementedwith 10% fetal bovine serum (Sigma) was preliminarily added to eachwell. By the calcium phosphate method (Graham L., et al., Virology, Vol.52, p. 456, 1973, Naoko Arai, Gene Introduction andExpression/Analytical Method, p. 13-15, 1994), the cell was transientlycotransfected with the GAL-PPARγ (0.15 μg/well) prepared in Example 5(1)and a reporter construct with the GAL4 binding region being repeatedlyarranged in a number of eight upstream the luciferase gene (RE×8-Luci;Shimokawa, et al., International Publication No. WO 99/04815) (0.8μg/well) together with pcDNA-AOP2 (0.05-0.2 μg/well). After 2 mM of thePPARγ agonist GW7282 or a test compound was added to the culture mediumfor 48-hour culturing, the culture medium was discarded and the cellswere rinsed with a phosphate buffered saline (abbreviated as PBShereinafter). Subsequently, 0.4 ml of a cell lysis solution (100 mMcalcium phosphate, pH 7.8, 0.2% Triton X-100) was added to each well, tomake the cell lytic. To 100 μl of the cell solution was added 100 μl ofa luciferase substrate solution (Picker Gene), to assay the emission oflight for 10 seconds using a chemiluminescence assay apparatus of TypeAB-2100 (ATTO). Plasmid pCH110 (Amersham Pharmacia Biotech) with theluciferase reporter gene and concurrently with the β-galactosidaseexpression gene was cotransfected at 0.4 μg/well into the cell, to assaythe β-galactosidase activity using a detection kit of P-galactosidaseactivity, namely Galacto-Light Plus™ system (Applied BioSystems) toexpress the activity in numerical figure. As the numerical figure wasdefined as the transfection efficiency of the introduced gene, theluciferase activity was corrected per each well.

As the results of the experiment, it was observed that the transcriptioninduction activity of PPARγ in an agonist-dependent manner wasdistinctly inhibited, in a manner depending on the dose of AOP2expression plasmid transfected into the cell (FIG. 5). This apparentlyindicates that the occurrence of the ligand-dependent interactionbetween PPARγ and ECHLP suppressed the transcription induction activityof PPARγ.

Based on the fact and the results described above that AOP2 wasexpressed in tissues including kidney and the amount of the AOP2 proteinwas increased in the diabetic model mice, it was believed that theamount of AOP2 in the cells in the diabetic symptoms was increased sothat the following excess promotion of the PPARγ activity in specifictissues such as kidney caused the adverse action (edema).

Because AOP2 contains a peroxidase-like sequence within the molecule,AOP2 is called anti-oxidant protein 2 (GenBank Accession No.XM_(—)001415) due to the homology in amino acid sequence. However, areport tells that as an actual physiological activity thereof, AOP2functions as an acidic calcium-independent phospholipase A2 (Kim TS, etal., J. Biol. Chem., Vol.272, No.16, p.10981, 1997), while anotherreport tells that the gene locus of the Aop2 protein is the etiologicalgene of polycystic nephropathia in mouse (LTW4/Aop2; lakoubova OA, etal., Genomics, Vol. 42, No. 3, p. 474-478, 1997). As described above,apparently, AOP2 has an action different from the molecular functionspeculated on the basis of the structure of the amino acid sequence.Therefore, the essential physiological function has not yet beenidentified. The finding by the inventors that AOP2 binds to PPARγ in aligand-dependent manner and functions as a transcriptional cofactorthereof is a novel finding from the standpoint of the function of themolecule. The use of AOP2 enables discovering and eliminating PPARγagonists triggering edema.

(Example 9) Screening System for Compounds Selectively Activating theMain Action Via PPARγ

Based on the findings, a novel therapeutic agent of diabetes mellitus byameliorating glucose metabolism and thereby making contributions to therecovery from diabetic symptoms can be screened by screening a compoundinhibiting the interaction between ECHLP and PPARγ and the suppressionof the ligand-dependent transcription promoting ability of PPARγ withECHLP, which are detectable in the reporter assay system in Example 5. Atherapeutic agent of diabetes mellitus by making contributions to therecovery from diabetic symptoms with no occurrence of edema as theadverse action can be screened by screening a substance never triggeringthe interaction between AOP2 and PPARγ and the activation of theligand-dependent transcription promoting ability of PPARγ with AOP2,which are detectable in the reporter assay system in Example 8, amongthe test substances obtained thereby.

Test compounds can be screened in the assay system of the reporteractivity, which is absolutely the same as in Examples 5 and 8.Nonetheless, the following reporter assay system was constructed so asto efficiently screen a greater number of test compounds.

The detailed method was the same as shown in Example 5. Under conditionsinvolving the inhibition of the transcription activating ability ofPPARγ with ECHLP in the presence of a PPAR agonist, compounds inhibitingthe suppressive action of the transcription activating ability werescreened by making an excess amount of a test compound concurrentlyexist for competition. Specifically, the culture cell COS-1 was culturedto a 70-% confluent state in a 6-well culture plate containing theminimum essential culture medium DMEM supplemented with 10% fetal bovineserum. By the calcium phosphate method, the cell was transientlycotransfected with the GAL-PPARγ (0.15 μg/well) and RE×8-Luci (0.8μg/well) together with pcDNA-ECHLP (0.15 μg/well). Under the conditionthat GW7282 as a PPARγ agonist was added to the culture to a finalconcentration of 0.1 μM, a test compound (10-1.0 μM) was added to theculture medium for 48-hour culturing in their concurrent presencethereof. Subsequently, the cell was rinsed with PBS, to which the celllysis solution was added at 0.4 ml/well to each well, to make the celllytic. 100 μl of the solution was transferred into a 96-well plate.According to the method of Example 5, then, the luciferase activity andthe β-galactosidase activity were assayed to numerically express theactivation of PPARγ. Based on the suppression of the ligand-dependenttranscription induction ability of PPARγ (ratio of corrected luciferaseactivity value) via the ECHLP expression as observed under the conditioninvolving the presence of a low concentration of GW7282 (0.1 μM) addedas a PPARγ agonist, a compound inhibiting the transcription inductionability was screened under the condition of an excess amount of a testcompound added at 10 or 0.1 μM. The standard for screening a substanceinhibiting the suppression of the PPARγ transcription induction abilityvia ECHLP is preferably 10 μM or less, more preferably 1.0 μM or less onthe basis of the intensity of the inhibitory activity (IC50). In thisscreening system, the aforementioned compound GI-262570 at 10 μMpartially inhibited the suppression of the ligand (0.1 μMGW7282)-dependent PPARγ transcription induction ability with ECHLP (FIG.6 a). Alternatively, the compound GL-100085 even at 10 μM neverinhibited the suppression of the transcription induction ability, whileGI-262570 was highly specific to the main action through PPARγ. Thus,GL-100085 could be actually selected as a compound with a low degree ofthe main action.

Continuously, the individual compounds (10-1.0 μN) selected in thescreening system were singly added to a screening system wherepcDNA-ECHLP in the above screening system was substituted withpcDNA-AOP2 (0.15 μg/well), so as to examine whether or not the promotionof the transcription induction ability of PPARγ with AOP2 in a mannerdepending on the test compound existed, by assaying the luciferaseactivity corrected in the same manner as described above. In thescreening system, it was confirmed that the compounds GW7282 andGL-100085 at 1.0 to 10 μM promoted the transcription induction abilityof PPARγ in the presence of AOP2 about 4 to 5-fold or 4- to 6-fold in amanner depending on the presence of each of the compounds. Meanwhile,the compound GI-262570 at any concentration of 1.0 μM and 10 μM promotedthe transcription inducing ability, only about 3.5-fold (FIG. 6B). Thisenabled actual selection of GL-100085 as a compound highly specific tothe adverse action through PPARγ in particular and GI-262570 as acompound with a relatively low specificity to the triggering of theadverse action.

(Example 10) Comparison of Expression Levels of FLJ13111 in Tissues

Using the Primers of SEQ ID NOS: 18 and 19, a cDNA fragment encodingFLJ13111 was amplified from the human cDNA library (Clontech) by PCR[DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo Co., Ltd.) wasused for treatment at 98° C. (one minute) and subsequent treatment witha cycle of 98° C. (5 seconds)/55° C. (30 seconds) and 72° C. (3 minutes)35 times], which was then detected by agarose gel electrophoresis.Consequently, FLJ13111 was distinctly expressed in muscle and liverwhere the PPARγ action could be observed among the main organs andadditionally in mammary gland, lung, placenta, ovary, lymphocyte, andleukocyte. In kidney responsible for the triggering of edema with aPPARγ ligand, however, almost no expression was observed. This supportedthat FLJ13111 was a transcriptional cofactor of PPARγ.

(Example 11) Detection of the Regulatory Action of FLJ13111 on theLigand-Dependent Transcription Induction Ability of PPARγ

The results of the yeast two-hybrid analysis indicated that FLJ13111interacted with PPARγ via the ligands thereof. Therefore, it wasexamined by the reporter assay using the culture cell COS-1 what kind ofinfluences FLJ13111 had on the transcription induction activity ofPPARγ.

(1) Preparation of Plasmid pcDNA-FLJ13111 for Animal Cell Expression

Using the primers of SEQ ID NOS: 18 and 19, a cDNA fragment of 897 bpconsisting of SEQ ID NO: 16 and encoding FLJ13111 was obtained from thehuman liver cDNA library (Clontech) by PCR [DNA polymerase (Pyrobest DNApolymerase; Takara Shuzo Co., Ltd.) was used for treatment at 98° C.(one minute) and for subsequent 35-time repetition of a cycle of 98° C.(5 seconds)/55° C. (30 seconds) and 72° C. (3 minutes)]. By the TOPOcloning method (Invitrogen), this cDNA fragment was inserted inpCDNA3.1/V5-His-TOPO vector (Invitrogen) by in vitro recombination, forpreparing a plasmid pcDNA-FLJ13111 for animal cell expression. Herein,no termination codon was inserted for the FLJ13111. A primer wasdesigned so that the vector-derived V5 epitope and the His tag might befused at the C terminus.

(2) Detection of the Regulatory Action of FLJ13111 on theLigand-Dependent Transcription Induction Ability of PPARγ

By the same method as in Example 5(3), pcDNA-ECHLP was substituted withpcDNA-FLJ13111 to thereby prepare a system for assaying the FLJ13111action on the ligand-dependent transcription induction ability of PPARγby the reporter assay. Additionally, 1 mM rosiglitazone was used as aPPARγ agonist. Rosiglitazone was added to assay the luciferase activity.Consequently, it was observed that the agonist-dependent transcriptioninduction activity of PPARγ was promoted in a manner depending on thedose of the FLJ13111-expressing plasmid having transfected the cell(FIG. 7). This apparently showed that the occurrence of theagonist-dependent interaction between PPARγ and FLJ13111 promoted thetranscription induction activity of PPARγ.

This fact and almost no expression of FLJ13111 in kidney responsible forthe triggering of edema with a PPARγ ligand suggested that the promotionof the PPARγ activity by FLJ13111 enhanced not the adverse action butthe main action.

FLJ13111 is a protein with unknown function. Although the presence of anuclear targeting sequence having been suggested to exist in cellnucleus and the presence of a possible N-glycosylation site, both withinthe molecule, could be speculated from the amino acid sequence, noadditional information has existed yet suggesting the molecular functionof FLJ13111 based on the amino acid sequence and structure. The findingof the inventors that FLJ13111 binds to PPARγ in a ligand-dependentmanner and functions as its transcriptional cofactor is a novel findingabout the function of the molecule. Utilizing the FLJ13111 can lead tothe discovery of an agonist selective to the main action through PPARγ.

(Example 12) Detection of the Ligand Selective Action of FLJ13111 ViaPPARγ and Screening System for Compounds Selectively Activating the MainAction Through PPARγ

Based on the findings, a novel therapeutic agent of diabetic mellitus byameliorating glucose metabolism and making contributions to the recoveryfrom diabetic symptoms can be screened, by screening a compoundpromoting the FLJ13111 action of activating the ligand-dependenttranscription promoting ability of PPARγ, which can be detected with thereporter assay system in Example 11. From the test compounds thusobtained, additionally, a substance inhibiting the interaction of AOP2and PPARγ and the activation of AOP2 on the ligand-dependenttranscription promoting ability of PPARγ as detectable with the reporterassay in Example 8 can be screened, to screen for a therapeutic agent ofdiabetes mellitus making contributions to the recovery from diabeticsymptoms with no occurrence of edema as the adverse action.

With absolutely the same reporter activity assay system as in Example11, specifically, test compounds can be screened. For efficientscreening of a greater number of test compounds, the reporter assay wasdone under the following preset conditions. Plasmid pCH110 (0.4 μg/well)with GAL-PPARγ (0.15 μg/well), a reporter construct (RE×8-Luci; 0.8μg/well) and the β-galactosidase expression gene was transientlycotransfected in COS-1 cell together with pcDNA-FLJ13111 (0.1 μg/well).To the resulting mixture in the culture medium was added a test compoundto a final concentration of 10-0.1 μM for culturing for 48 hours, toassay the luciferase activity and the β-galactosidase activity andexpress the activation of PPARγ in numerical figure. Details of thetransfection method and the method for assaying luciferase underconditions except for the conditions described above followed Examples 5and 9. The test compounds were screened using as a marker the promotionof the transcription induction ability of PPARγ via FLJ13111 expression(ratio of corrected luciferase activity values) under individualconditions with added test compounds or without any test compound added.The standard for screening a substance promoting the PPARγ transcriptioninduction ability with FLJ13111 was based on the effective concentration(ED50) being preferably 10 μM or less, more preferably 1.0 μM or less.In the screening system, rosiglitazone and pioglitazone both at 1 μMpromoted the PPARγ transcription induction ability with FLJ13111.Meanwhile, the compound GL-100085 even at 10 μM did not promote thetranscription induction ability; rosiglitazone and pioglitazone werehighly specific to the main action through PPARγ; and GL-100085 couldactually be selected as a compound with a low level of the main action.

(Example 13) Assaying FLJ13111 Expression Level in Normal Mouse andDiabetic Model Mouse

Based on the finding, it was deduced that the interaction between theFLJ13111 protein and PPARγ might be involved in the amelioration ofglucose metabolism as the main action via a PPARγ agonist. Thus, themessenger RNA (mRNA) expression level of the mouse ortholog gene in theFLJ13111 gene was assayed in the muscle of the two types of diabeticmodel mice in Example 4, namely KKA^(y)/Ta and C57BL/KsJ-db/db, and wascompared with the level in normal individual mice C57BL/6J andC57BL/KsJ-m+/m+. As to the expression level of the gene, the expressionlevel of the FLJ13111 gene in accordance with the invention was assayedand corrected on the basis of the expression level of the glyceraldehyde3-phosphate dehydrogenase (G3PDH) gene concurrently assayed. As theassay system, PRISM™ 7700 Sequence Detection System and SYBR Green PCRMaster Mix (Applied BioSystems) were used. The fluorescence intensity ofthe dye SYBR Green I incorporated by double-stranded DNA amplified byPCR was subjected to real-time detection and assaying, to determine theexpression level of the intended gene.

Specifically, the following procedures were used for the assay.

(1) Resection of Mouse Tissues and Extraction of mRNA

By the same methods as in Example 4, the tissues and mRNA were prepared.

(2) Synthetic Preparation of Single-Stranded cDNA

Reverse transcription from total RNA to single-stranded DNA was done ina system of 20μl, using individually (1) 1 μg of RNA prepared above in(1) (muscle from mice of age 15 or 12 weeks old) and areverse-transcription kit (Advantage™ RT-for-PCR kit; Clontech). Afterreverse-transcription, 180 μl of aseptic water was added to theresulting DNA, for storage at −20° C.

(3) Preparation of PCR Primer

Four oligonucleotides (SEQ ID NOS: 20 through 24) were designed as thePCR primers described in the item (4). A combination of SEQ ID NOS: 20and 21 was used for the FLJ13111 gene, while a combination of SEQ IDNOS: 22 and 23 was used for the G3PDH gene.

(4) Assaying Gene Expression Level

The real-time assay of the PCR amplification with PRISM™ 7700 SequenceDetection System was done in a 25-μl system according to the instructionmanual. For each system, 5 μl of single-stranded cDNA, 12.5 μl of the2×SYBR Green reagent and 7.5 pmol of each of the primers were used.Herein, the cDNA prepared in (1) was used; the cDNA was diluted 30-foldfor G3PDH; and the cDNA was diluted 10-fold for FLJ13111. For preparinga standard curve, an appropriate dilution of the murine genome DNA at0.1 μg/μl (Clontech) was used at a volume of 5 μl, in place of thesingle-stranded cDNA. PCR was done at 50° C. for 10 minutes andcontinuously at 95° C. for 10 minutes, and subsequently by repeating atwo-step process consisting of 95° C. for 15 seconds and 60° C. for 60seconds 45 times.

The expression level of the murine FLJ13111 gene in each sample wascorrected on the basis of the expression level of the G3PDH geneaccording to the following formula.[Corrected FLJ13111 expression level]=[Expression level (raw data) ofFLJ13111 gene]/=[Expression level (raw data) of G3PDH]

For comparison of the expression level, the relative value thereof isshown in FIG. 8, when the expression level of C57BL/6J mouse was definedas 100. As shown in FIG. 8, apparently, the expression of the FLJ13111gene was lowered markedly in the muscle of the diabetic model mice.Thus, it is considered that the reduction of the FLJ13111 expressionlevel in the muscle triggers insulin resistance. Based on thosedescribed above, it is concluded that FLJ13111 is largely involved ininsulin resistance.

Additionally, the results in this Example apparently indicated thatdiabetic symptoms can be diagnosed by assaying the expression level ofFLJ13111.

(Example 14) Identification of FLJ13111 Promoter Sequence, and Screening

System for a Compound Selectively Activating the Main action, utilizingthe transcription induction activity of the sequence Based on thefinding in Example 11 above, apparently, the increase of the existingFLJ13111 enhances the action of a PPARγ ligand with a high effect on thetriggering of the main action. Based on the fact, the possibility ofameliorating insulin resistance is anticipated by positively adjustingthe FLJ13111 expression level from the FLJ13111 gene. However, not anypromoter sequence responsible for the regulation of the expression ofthe FLJ13111 is known. Therefore, attempts were made to obtain anFLJ13111 promoter sequence. First, a pair of primers of SEQ ID NOS: 24and 25 were designed. Using these primers under the same PCR conditionsas described in Example 11 (1), the amplification of the FLJ13111promoter sequence was attempted. Finally, success was made in theamplification of a CDNA fragment of about 1.8 kbp. By the same method asin the Example, the nucleotide sequence of the fragment was determined.Thus, it was found that the fragment was the polynucleotide of SEQ IDNO: 26, containing a part of the coding sequence of the FLJ13111 gene atthe 3′ terminus. It was determined by the following method whether ornot the polynucleotide sequence had a promoter activity regulating theexpression of FLJ13111. Inserting the nucleotide at the multicloningsite of pGL3-Basic Vector (Promega) as a luciferase reporter vector,using restriction enzymes BgIII and HindIII, a reporter plasmid namedpGL3-FLJ13111p was prepared. The plasmid was transfected in COS-1 cell.By comparison with a case of transfection in pGL3-Basic Vector (vacantvector) never carrying the polynucleotide, the activity of thepolynucleotide as promoter to induce the expression was assayed, usingthe luciferase activity as a marker. The correction of the transfectionefficiency into cells and the luciferase assay were precisely the sameas used in the method described in Example 5(3). Consequently, asignificant promoter activity depending on the presence of thepolynucleotide was confirmed as shown in FIG. 9. Further, it wasrevealed that the promoter activity was activated when pioglitazone (0.1μM) as a PPARγ ligand was added to the transfected cell. At thisexperiment, additionally, the co-transfection with pcDNA-FLJ13111 as theFLJ13111 expression plasmid lowered the promoter activity of thepolynucleotide as shown in FIG. 9. These facts show that the clonedpolynucleotide sequence contained the promoter sequence regulating theFLJ13111 expression, and the promoter was positively regulated withPPARγ ligands reducing insulin resistance such as pioglitazone and wasnegatively regulated with FLJ13111 itself. Based on this, it is deducedthat not only FLJ13111 activates the activity of PPARγ via a ligand butalso the expression level of FLJ13111 per se is activated with a PPARγligand known to have an effect of reducing insulin resistance, both ofwhich synergistically act for reducing insulin resistance.

Based on the findings, the assay of the FLJ13111 promoter in the Examplecan be utilized for screening a PPARγ ligand or a drug amelioratinginsulin resistance with no use of the PPARγ protein.

(Example 15) Assaying Adipocyte Differentiation in Cells ExpressingECHLP Excessively

As described above, it was shown that the ECHLP protein bound to PPARγin a manner depending on the presence of a PPARγ ligand to suppress thetranscription induction activity of PPARγ. Because the expression levelof ECHLP is increased in diabetic symptoms, further, the excessexpression thereof triggers insulin resistance via the suppression ofthe PPARγ activity to cause Type 2 diabetes mellitus. Meanwhile, it isknown that PPARγ promotes the adipocyte differentiation by the inductionof the transcription activity depending on the ligand, so thatdifferentiated adipocytes incorporate blood glucose and thereby, glucosemetabolism is ameliorated and insulin resistance is reduced. It wasexamined at the following experiments whether or not overexpression ofECHLP in cells had an actual influence on the adipocyte differentiation,which had a relation with insulin resistance.

(1) Establishment of L1 Cell Expressing ECHLP Excessively

So as to recombine ECHLP with the FLAG sequence consisting of DYKDDDDKbeing added at the C terminus in a retrovirus vector pCLNCX(Immunogenetics), a BamHI-NotI fragment of about 1 kb was prepared fromthe pcDNA-ECHLP plasmid, using restriction enzymes. So as to prepare aDNA fragment including a NotI site-FLAG sequence-XbaI site, further, twosynthetic oligo DNAs of SEQ ID NOS: 27 and 28 were mixed together,heated and annealed to prepare double-stranded DNA fragments. These DNAfragments were recombined together at the BamHI and XbaI sites ofpCLNCX, to prepare a pCLNCX-ECHLP-Flag vector. The pCLNCX-ECHLP-FLAGvector and the pCL-Eco vector (Immunogen) were both introduced into the293 cell by the calcium phosphate method for the transfection. 24 and 48hours after the transduction, the recombinant virus in the culturesupernatant was recovered. The virus was diluted 2-fold with a freshcell culture broth [Minimum essential culture medium DMEM (Gibco)] notyet used, to which polybrene (Sigma) was added to a final concentrationof 8 μg/ml, to make the virus infect a murine cultured precursoradipocyte 3T3-L1 (ATCC). 48 hours after the infection and thereafter,the virus-infected cell was screened for with 1.5 mg/ml G418 (Nakarai),to establish an L1 cell expressing ECHLP-FLAG in a stable fashion. As acontrol, a cell infected with pCLNCX vector (vacant vector) was alsoprepared. The expression of ECHLP-FLAG in the established cell wasconfirmed by Western blotting using anti-FLAG M2 antibody (Sigma).Specifically, 10 μl of 2×SDS sample buffer (125 mM Tris-HCl, pH 6.8, 3%sodium laurylsulfate, 20% glycerin, 0.14 M β-mercaptoethanol, 0.02%bromophenol blue) was added to. 10 μl of the solution of the lytic cellexpressing ECHLP-FLAG, for treatment at 100° C. for 2 minutes, for 10%SDS polyacrylamide gel electrophoresis, to separate the proteincontained in the sample. Using a semi-dry type blotting apparatus(BioRad), the protein in the polyacrylamide was transferred onto anitrocellulose membrane, to detect the ECHLP protein on thenitrocellulose by general procedures according to Western blotting. Amonoclonal antibody recognizing the FLAG epitope fused at the C terminusof ECHLP was used as a primary antibody, while the rabbit IgG-HRP fusedantibody (BioRad) was used as a secondary antibody. Consequently, it wasconfirmed that the protein representing the ECHLP-FLAG fused protein wasdetected in a manner depending on the cell introduction of theECHLP-FLAG expression vector.

(2) Adipocyte Differentiation with Pioglitazone

The vacant vector-infected L1 cell or the ECHLP-infected L1 cellestablished by the method was cultured at 10⁴ cells/well in a 96-wellplate. 48 hours later and thereafter, the cell was differentiated andinduced into adipocyte, using insulin (1 μg/ml) and pioglitazone (0.1-3μM). Concerning the degree of the adipocyte differentiation, the contentof triglyceride incorporated in the cell was used as a marker. The cellon day 7 after the start of the induction of the differentiation wasused to assay the content of triglyceride.

(3) Assaying Intracellular Triglyceride

The cell in the 2 wells was dissolved in 40 μl of 0.1% SDS solution, towhich 1 ml of a triglyceride assay reagent (Triglyceride G-test Wako,Wako Pure Chemical Industries Ltd.) was added, for heating at 37° C. for10 minutes. The absorbance of the reaction solution at a wavelength of505 nm (OD505) was measured. As shown in FIG. 10, consequently, it wasobserved that the intracellular triglyceride increased in the controlcell (vacant vector-infected L1 cell) in a manner dependent on the doseof pioglitazone (0.1-3 μM), indicating the adipocyte differentiation.Alternatively, the triglyceride increase induced by pioglitazone (0.1-3μM) at any of the dose in the cell expressing ECHLP excessively(ECHLP-infected L1 cell) was suppressed to 43 to 57% of the increment inthe control cell.

The suppression of the adipocyte differentiation decreases the totalamount of glucose incorporation induced by adipocyte. Thus, the aboveresults clearly show that the excess expression of ECHLP suppresses theadipocyte differentiation, so that it works as a causative factor oftype 2 diabetes mellitus.

(Example 16) Identification of Ligand Selectively Inducing the BindingBetween FLJ13111 and PPARγ

Screening by the same reporter assay as shown above in Example 12 wasdone. Consequently, a compound XF promoting the transcription inductionactivity of PPARγ was obtained (FIG. 11). It was shown that the titerthereof at 10 μM was approximately comparable to 0.1 μM of pioglitazone.Further, it was found that the promotion action of the compound XF onthe transcription activating ability of PPARγ was activated by excessexpression of FLJ13111 (0.1 μg/well) in the same fashion as in the caseof pioglitazone.

In the system for assaying the ligand-dependent binding of PPARγ toFLJ13111 by the yeast two-hybrid method as shown in Example 2, thecompound XF was experimentally used under the same conditions in placeof GW7282. It was found that the compound XF never induced the bindingof PPARγ with the aforementioned proteins SRC-1, ECHLP and AOP2, butinduced only the binding of PPARγ with FLJ13111.

(Example 17) Assaying the Expression Level of Sodium-Potassium ATPasewith FLJ13111-Selective PPARγ Ligand

Edema caused by PPARγ ligand is induced by the increase of circulatingplasma volume, which is known to occur in relation with the increase ofthe expression level of the sodium-potassium ATPase in renal cell.Therefore, it was examined whether or not the compound XF influenced theexpression level of the sodium-potassium ATPase in renal cell, which hasa relation with triggering edema.

Specifically, feline renal epithelial cell MDCK was cultured at 1.5×10⁵cells/well in a 24-well culture plate, using the minimum essentialculture medium DMEM (Gibco) supplemented with 10% fetal bovine serum(Sigma) at 37° C. for 48 hours. The solvent (dimethylsulfoxide) alone orpioglitazone (to a final concentration of 0.1 to 10 μM) or the testcompound XF (to a final concentration of 0.1 to 10 μM) was added to theliquid culture, for culturing for another 6 hours. After the cell wasrinsed two times with 1 ml of an assay buffer (3 mM MgSO₄, 3 mM Na₂HPO₄,10 mM Tris-HCl, 250 mM sucrose), 200 μl of an assay buffer containing³H-ouabain (74 Bq/μl; Amersham Bioscience) and 2 μM ouabain was added tothe cell, which was then left to stand at 37° C. for 2 hours. Theradioactivity bound under the conditions was defined as total binding.For assaying non-specific binding, additionally, ³H-ouabain (74 Bq/μl)and 1 mM ouabain were used. After removing the reaction solution underaspiration, the cell was rinsed three times with 1 ml of an ice-coldassay buffer, to solubilize the cell with an aqueous 0.5 N NaOH solution(250 μl). After the resulting solution was made neutral with an equalvolume of aqueous 0.5N HCl solution, 5 ml of a liquid scintillator wasadded to count the radioactivity with a liquid scintillation counter.The specific binding with ³H-ouabain was determined by subtracting thenon-specific binding value from the total binding value, to determinethe expression level of the sodium-potassium ATPase.

As shown in FIG. 12, the results are that pioglitazone added at 0.1 μMexerted a significant action of enhancing the expression level of thesodium-potassium ATPase, compared with the control cell with the solventadded alone. Meanwhile, the compound XF never enhanced the expressionlevel of the sodium-potassium ATPase even when the compound XF was addedat a 10-μM concentration at which the XF showed almost the same effecton the PPARγ transcription activation as pioglitazone did. In otherwords, it was revealed that the compound XF as an FLJ13111-selectivePPARγ ligand never induced the increase of the expression level of thesodium-potassium ATPase triggering edema. Thus, it was shown that thecompound XF was never involved in triggering edema.

(Example 18) Assay of the Differentiation Ability into Adipocyte ViaFLJ13111-Selective PPARγ Ligand

The same method as in Example 15 was used to examine whether or not theaddition of the compound XF had an influence on the adipocytedifferentiation, which had a relation with the reduction of insulinresistance. Specifically, the compound XF was added (1.0-10.0 μM) to theprecursor adipocyte 3T3-L1 (ATCC) cultured in mouse. Using the amount oftriglyceride on day 7 in the cell as a marker, the differentiation levelinto adipocyte was measured. Consequently, it was observed that thecompound XF increased the triglyceride amount by about 20%, comparedwith the cell with a solvent added alone.

The promotion of the adipocyte differentiation increases the totalglucose uptake for which adipocyte is responsible, so that insulinresistance is ameliorated. Thus, the results above indicate that thecompound XF has an action of ameliorating insulin resistance with noinduction of edema.

The results described above clearly show that FLJ13111 can be used forscreening a compound selectively having the main action but nevercausing the adverse action, namely a drug ameliorating insulinresistance.

Industrial Applicability

By the yeast two-hybrid screening method to be carried out in thepresence of ligands in accordance with the invention, a proteininteractive with PPARγ in a ligand-dependent manner working as a usefultool for screening a drug ameliorating insulin resistance without theadverse action can be screened. The use of the main actionligand-dependent PPAR-binding molecule ECHLP, the main actionligand-selective PPARγ-interactive factor FLJ13111, and the adverseaction ligand-dependent PPAR-binding molecule AOP2 as obtained by themethods enables the identification and screening of a compound havingselectively the main action but never causing the adverse action. Thesubstance selected with the screening system is useful as a candidatesubstance as a drug for ameliorating insulin resistance.

Sequence Listing Free Text

In the numerical title [223] in the Sequence Listing below, the[Artificial Sequence] is described. Specifically, individual nucleotidesequences of SEQ ID NOS: 9, 10, 11, 13, 24, 25, 27 and 28 in theSequence Listing are primer sequences artificially preparedsynthetically.

The invention has been described above with reference to the specificembodiments. Variations and modifications thereof obvious to personsskilled in the art are also encompassed within the scope of theinvention.

1. A method for screening a protein interactive with PPARγ in aligand-dependent manner, utilizing a yeast two-hybrid system in thepresence of a PPAR ligand with a high potency of triggering the actionameliorating glucose metabolism, wherein a polynucleotide encoding aregion containing at least the position 204 to position 505 of the PPARγprotein represented by SEQ ID NO: 2 is used as bait and a cDNA libraryis used as prey.
 2. A method for screening a protein interactive withPPARγ in a ligand-dependent manner, utilizing a yeast two-hybrid systemin the presence of a PPAR ligand with a high potency of triggeringedema, wherein a polynucleotide encoding a region containing at leastthe position 204 to position 505 of the PPARγ protein represented by SEQID NO: 2 is used as bait and a cDNA library is used as prey.
 3. A celltransformed by i) a polynucleotide encoding a polypeptide consisting ofan amino acid sequence of SEQ ID NO: 4 or a polynucleotide encoding apolypeptide comprising an amino acid sequence represented by SEQ ID NO:4 wherein 1 to 10 amino acids therein are deleted, substituted and/orinserted and also interacting with PPAR in a ligand-dependent manner,ii) a gene encoding a fusion protein comprising at least the ligandbinding region of the PPAR protein represented by SEQ ID NO: 2 or 6 andthe DNA binding region of a transcription factor, and iii) a reportergene fused to a response element to which said DNA binding region of thetranscription factor is capable of binding; or a cell transformed by i)a polynucleotide encoding a polypeptide consisting of an amino acidsequence of SEQ ID NO: 4 or a polynucleotide encoding a polypeptidecomprising an amino acid sequence represented by SEQ ID NO: 4 wherein 1to 10 amino acids therein are deleted, substituted and/or inserted andadditionally interacting with PPAR in a ligand-dependent manner and ii)a reporter gene fused to a response element to which the DNA bindingregion of the PPAR protein represented by SEQ ID NO: 2 or 6 is capableof binding, said cell expressing a) a polypeptide consisting of an aminoacid sequence of SEQ ID NO: 4 or a polypeptide comprising an amino acidsequence represented by SEQ ID NO: 4 wherein 1 to 10 amino acids thereinare deleted, substituted and/or inserted and interacting with PPAR in aligand-dependent manner and b) the PPAR protein represented by SEQ IDNO: 2 or
 6. 4. A cell transformed by i) a polynucleotide encoding apolypeptide consisting of an amino acid sequence of SEQ ID NO: 8 or applynucleotide encoding a polypeptide comprising an amino acid sequencerepresented by SEQ ID NO: 8 wherein 1 to 10 amino acids therein aredeleted, substituted and/or inserted and additionally interacting withPPAR in a ligand-dependent manner, ii) a gene encoding a fusion proteincomprising at least the ligand binding region of the PPAR proteinrepresented by SEQ ID NO: 2 or 6 and the DNA binding region of atranscription factor, and iii) a reporter gene fused to a responseelement to which said DNA binding region of the transcription factor iscapable of binding, or a cell transformed by i) a polynucleotideencoding a polypeptide consisting of an amino acid sequence of SEQ IDNO: 8 or a polynucleotide encoding a polypeptide comprising an aminoacid sequence represented by SEQ ID NO: 8 wherein 1 to 10 amino acidstherein are deleted, substituted and/or inserted and additionallyinteracting with PPAR in a ligand-dependent manner and ii) a reportergene fused to a response element to which the PPAR protein representedby SEQ ID NO: 2 or 6 is capable of binding, said cell expressing a) apolypeptide consisting of an amino acid sequence of SEQ ID NO: 8 or apolypeptide comprising an amino acid sequence represented by SEQ ID NO:8 wherein 1 to 10 amino acids therein are deleted, substituted and/orinserted and interacting with PPAR in a ligand-dependent manner, and b)the PPAR protein represented by SEQ ID NO: 2 or
 6. 5. A cell accordingto claim 3 or 4, wherein the transcription factor is the GAL4 protein ofyeast.
 6. A cell according to claim 3 or 4, wherein the reporter gene isluciferase gene.
 7. A method for detecting whether or not a testsubstance promotes the action of ameliorating glucose metabolism viaPPAR, comprising i) a step of allowing a cell according to claim 3, aPPAR ligand and a test substance in contact with each other, and ii) astep of analyzing the change of the ligand-dependent interaction or thechange of the transcriptional activity induced by ligand-activated PPAR,using the expression of a reporter gene as a marker.
 8. A method forscreening a drug ameliorating insulin resistance, comprising i) a stepof allowing a cell according to claim 3, a PPAR ligand and a testsubstance in contact with each other, and ii) a step of analyzing thechange of the ligand-dependent interaction or the change of thetranscriptional activity induced by ligand-activated PPAR, using theexpression of a reporter gene as a marker.
 9. A method for screeningaccording to claim 8, wherein the drug ameliorating insulin resistanceis a drug ameliorating glucose metabolism.
 10. A method for detectingwhether or not a test substance promotes the activity triggering edemavia PPAR, comprising i) a step of allowing a test substance in contactwith a cell according to claim 4, and ii) a step of analyzing the changeof the interaction due to the test substance or the change of thetranscriptional activity induced via PPAR due to the test substanceusing the expression of a reporter gene as a marker.
 11. A method forscreening a drug ameliorating insulin resistance with no activity oftriggering edema, comprising i) a step of allowing a test substance incontact with a cell according to claim 4, ii) a step of analyzing thechange of the interaction due to the test substance or the change of thetranscriptional activity induced via PPAR due to the test substance,using the expression of a reporter gene as a marker; and iii) a step ofselecting a test substance not enhancing the reporter activity.
 12. Amethod for screening according to claim 11, wherein the drugameliorating insulin resistance is a drug ameliorating glucosemetabolism.
 13. A cell transformed by i) a polynucleotide encoding apolypeptide consisting of an amino acid sequence of SEQ ID NO: 17 or apolynucleotide encoding a polypeptide comprising an amino acid sequencerepresented by SEQ ID NO: 17 wherein 1 to 10 amino acids therein aredeleted, substituted and/or inserted and also interacting with PPAR in aligand-dependent manner, ii) a gene encoding a fusion protein comprisingat least the ligand binding region of the PPAR protein represented bySEQ ID NO: 2 or 6 and the DNA binding region of a transcription factor,and iii) a reporter gene fused to a response element to which said DNAbinding region of the transcription factor is capable of. binding; or acell transformed by i) a polynucleotide encoding a polypeptideconsisting of an amino acid sequence of SEQ ID NO: 17 or apolynucleotide encoding a polypeptide comprising an amino acid sequencerepresented by SEQ ID NO: 17 wherein 1 to 10 amino acids therein aredeleted, substituted and/or inserted and additionally interacting withPPAR in a ligand-dependent manner and ii) a reporter gene fused to aresponse element to which the PPAR protein represented by SEQ ID NO: 2or 6 is capable of binding, said cell expressing a) a polypeptideconsisting of an amino acid sequence of SEQ ID NO: 17 or a polypeptidecomprising an amino acid sequence represented by SEQ ID NO: 17 wherein 1to 10 amino acids therein are deleted, substituted and/or inserted andinteracting with PPAR in a ligand-dependent manner, and b) the PPARprotein represented by SEQ ID NO: 2 or
 6. 14. A method for detectingwhether or not a test substance promotes the action of amelioratingglucose metabolism via PPAR, comprising i) a step of allowing a testsubstance in contact with a cell according to claim 13, and ii) a stepof analyzing the change of the interaction due to the test substance orthe change of the transcriptional activity induced via PPAR due to thetest substance, using the expression of a reporter gene as a marker. 15.A method for screening a drug ameliorating insulin resistance,comprising i) a step of allowing a cell according to claim 13 in contactwith a test substance, and ii) a step of analyzing the change of theinteraction due to the test substance or the change of thetranscriptional activity induced via PPAR due to the test substance,using the expression of a reporter gene as a marker.
 16. A method forscreening according to claim 15, wherein the drug ameliorating insulinresistance is a drug ameliorating glucose metabolism.
 17. A method forscreening a drug ameliorating insulin resistance, comprising i) a stepof allowing a test substance in contact with a cell transformed with areporter gene fused to a polynucleotide consisting of a nucleotidesequence of SEQ ID NO: 26 or a polynucleotide comprising a nucleotidesequence represented by SEQ ID NO: 26 wherein 1 to 10 bases therein aredeleted, substituted and/or inserted and also having a transcriptionpromoter activity, and ii) a step of analyzing the change of theactivity for transcriptional induction due to the test substance, usingthe expression of a reporter gene as a marker.
 18. A method forscreening according to claim 17, wherein the reporter gene is theluciferase gene.
 19. A method for producing a pharmaceutical compositionfor ameliorating insulin resistance, comprising a screening step using ascreening method according to claim 8, 11, 15 and/or 17 and aformulation step using a substance obtained by the screening.